Methods for treating autoimmune and chronic inflammatory conditions using antagonists of CD30 or CD30L

ABSTRACT

The invention provides methods of treating autoimmune and chronic inflammatory conditions by administering agents that hinder the CD30/CD30L interaction, combination treatments, and methods of treating conditions resistant to treatment with TNFα inhibitors by administering agents that inhibit signal transduction by CD30 or IL-1. Included also are treatments involving concurrently administering agents that block the CD30/CD30L interaction and agents that antagonize the IL-4/IL-4R interaction. Additionally provided is an animal model for screening candidate agents for their efficacy in treating conditions that are resistant to treatment with TNFα inhibitors.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a divisional of U.S. patent application Ser.No. 09/921,667 filed on Aug. 3, 2001, now allowed, which claims thebenefit of priority from U.S. patent application Ser. No. 60/224,079,filed Aug. 8, 2000.

FIELD OF THE INVENTION

[0002] This invention relates generally to methods for treatingautoimmune and inflammatory disorders that involve administering agentsthat block the interaction between CD30 and CD30L or IL-1 and IL-1R1,and also provides an animal model for testing the ability of a compoundto treat inflammatory conditions that respond poorly to treatment withTNFα inhibitors.

BACKGROUND OF THE INVENTION

[0003] CD30 and its ligand, CD30L, are membrane proteins of the TNFR andTNF ligand superfamilies, respectively, and are expressed on variouslymphoid and myeloid cells. CD30 was first described as an antigen onHodgkin's disease cells, and presently is widely used as a clinicalmarker for a number of hematologic malignancies (for review, see Horieand Watanabe, Immunol 10:457-470 (1998)). A naturally-occurring solubleform of the CD30 protein is found in human serum, and levels of thisprotein are elevated in a variety of pathological conditions includingviral infection, allergic and autoimmune conditions and neoplasticdiseases.

[0004] CD30 and CD30L are expressed on T cells, and appear to beinvolved in regulation of the immune system. Their expression on T cellsis activation-dependent. CD30 has been reported to be a specific markerof T_(H)2 type T cells (Romagnani, J Leukocyte Biol 57:726 (1995);Romagnani, J Clin Immunol 15:121-129 (1995)). The T_(H)1 and T_(H)2subsets of CD4⁺ T cells can be distinguished based on which cytokinesthey predominantly express. Though individual T cells may actuallysecrete mixtures of these two groups of cytokines, chronic immunereactions are often dominated by one type or the other of CD4⁺ T cells.T cells that produce T_(H)2 cytokines, which include IL-4, generally areconsidered to be mediators of allergic reactions. It has been suggestedthat the detection of circulating CD30⁺ T cells could serve as a markerfor T_(H)2-dominated allergic conditions such as atopic dermatitis(Yamamoto et al., Allergy 55:1011-18 (2000)); however, the correlationbetween CD30 expression and T_(H)2 phenotype has not held up over time(see, for example, Bengtsson et al, J Leukocyte Biol 58:683 (1996);Hamann et al., J Immunol 156:1387-91 (1996)). Based on experiments usinga mouse model of diabetes, it has been proposed also that CD30-mediatedsignaling may protect against autoimmune disease (Kurts et al., Nature398:341-344 (1999)). Others have reported that IL-4 upregulates, whereasIFNγ downregulates, the expression of CD30 on activated T cells(Nakamura et al., J Immunol 158:2090-98 (1997); Gilfillan et al., JImmunol 160:2180-87 (1998)).

[0005] The naturally-occurring ligand for CD30, CD30L, is a type IImembrane glycoprotein that binds specifically with CD30, thus triggeringCD30 to transmit a signal via its cytoplasmic domain. The isolation ofmouse and human cDNAs encoding CD30L is described in U.S. Pat. No.5,480,981. In addition to being expresed on activated T cells, CD30L isexpressed on monocytes/macrophages, granulocytes and a subset of B cells(see, for example, U.S. Pat. No. 5,480,981). CD30L has been reported toinduce murine B cell differentiation and the proliferation of activatedT cells in the presence of an anti-CD3 co-stimulus (see, for example,Smith et al., Cell 73:1349-1360 (1993)). Moreover, it has been reportedthat CD30L exhibits “reverse signaling,” that is, the cell surface CD30Lthat is expressed on neutrophils and peripheral blood T cells can beactivated by cross-linking to stimulate metabolic activities in thosecells (Wiley et al., J Immunol 157: 3235-39 (1996)).

[0006] There is a need to better understand the biological activities ofCD30 and CD30L, and to exploit this knowledge in the treatment of humandisease.

SUMMARY OF THE INVENTION

[0007] In accord with this invention, an agent capable of inhibiting thebinding of CD30 to CD30L is used for treating an autoimmune or chronicinflammatory condition, the method comprising administering the agent tothe patient according to a regimen of dose and frequency ofadministration that is adequate to induce a sustained improvement in atleast one indicator that reflects the severity of the patient'scondition. The improvement is considered to be sustained if the patientexhibits the improvement on at least two occasions separated by at leastone day. The agent may be formulated into a physiologically acceptablepharmaceutical preparation, which may be packaged with a written matterdescribing the foregoing use. Moreover, an inhibitor of the CD30/CD30Linteraction according to this invention may be administered concurrentlywith other treatments being used to treat the same disorder. In apreferred embodiment, the patient is a human.

[0008] In one of the embodiments of the invention, preferred agents foruse in treating an autoimmune or inflammatory condition include anantibody that is specific for CD30L, a non-agonistic antibody that isspecific for CD30, and a soluble CD30 polypeptide that comprises all orpart of the extracellular region of human CD30. The nucleotide and aminoacid sequences of human CD30 are shown in SEQ ID NO:6. Suitable CD30polypeptides for use as therapeutic agents include proteins thatcomprise amino acids 19-390 of SEQ ID NO:6, or a fragment thereof thatretains the ability to bind CD30L, including polypeptides having an atleast 80%, at least 85%, at least 90%, at least 95%, at least 97.5%, orat least 99%, and most preferably at least 99.5% sequence identity withamino acids 19-390 of SEQ ID NO:6. Such polypeptides if administered invivo are expected to bind with endogenous CD30L thereby interfering withthe interaction between endogenous CD30 and endogenous CD30L. Suchpolypeptides may if desired be conjugated with another moiety, usuallyanother protein, that promotes oligomerization. A moiety suitable forthis purpose is the Fc region from an immunoglobulin molecule. Agentsthat antagonize the CD30/CD30L interaction may be used to prepare apharmaceutical preparation to be administered in accord with the methodsof the invention, either alone or concurrently with other treatments.Such pharmaceutical preparations may be packaged with a written matterdescribing the aforedescribed uses.

[0009] The hereindescribed therapeutic agents that inhibit theCD30/CD30L interaction may be used to treat a variety of diseases,including various arthritic conditions. For example, rheumatoidarthritis may be treated with the CD30/CD30L antagonists that aredescribed above.

[0010] In one aspect of the invention, an agent capable of inhibitingthe interaction of CD30 and CD30L is used concurrently with a secondagent that is capable of antagonizing TNFα, IL-1α, IL-1β or IL-4.Medical disorders expected to be especially responsive to thesecombination treatments include multiple sclerosis, systemic sclerosis,acute inflammatory demyelinating polyneuropathy, acute motor axonalneuropathy, acute motor sensory axonal neuropathy, Fisher syndrome andsystemic lupus erythematosus. As an example, the foregoing medicaldisorders may be treated with an antibody specific for CD30L usedtogether with an antagonist of IL-4. The anti-CD30L antibody and theIL-4 antagonist are used for formulating pharmaceutical preparations forthis purpose, and may be packaged separately or together in one packagewith a written matter describing this use. Suitable IL-4 antagonists foruse in this method of treatment include but are not limited to anantibody specific for IL-4, an antibody specific for IL-4R and a solubleIL-4 receptor comprising amino acids 1-207 or 2-207 of SEQ ID NO:16. Inone of the preferred embodiments of the invention, a patient who issuffering from systemic lupus erythematosus, scleroderma or pemphigusvulgaris is treated concurrently with an inhibitor of the CD30/CD30Linteraction and an antibody specific for IL-4, an antibody specific forIL-4R or a soluble IL-4 receptor, wherein the receptor comprises aminoacids 1-207 or amino acids 2-207 of SEQ ID NO: 16.

[0011] In other aspects of the invention, provided are compounds,pharmaceutical preparations and methods of treatment for treating anautoimmune or chronic inflammatory condition that is resistant totreatment with an inhibitor of TNFα. This method comprises administeringto a patient in need thereof an agent that is capable of inhibiting thebinding of CD30 to CD30L or the binding of IL-1α or IL-10 to IL-R1,thereby blocking signal transduction by CD30 or IL-1. The agent isadministered according to a regimen of dose and frequency ofadministration that is adequate to induce a sustained improvement in atleast one indicator that reflects the severity of the patient'scondition, the improvement being considered sustained if the patientexhibits the improvement on at least two occasions separated by at leastone day. Agents suitable for use in such methods include an antibodythat is specific for CD30, CD30L, IL-1α, IL-1β or IL-R1, wherein theantibody may be a polyclonal antibody, a monoclonal antibody, ahumanized antibody or a human antibody. Agents to be used for thispurpose may be formulated into pharmaceutical preparations, which may bepackaged with a written matter describing such use. One exemplary agentfor this purpose is a soluble fragment of IL-1R2 that includes aminoacids 1-333 of SEQ ID NO:8, or a subportion thereof that is capable ofbinding with IL-1α or IL-1β, thereby blocking signal transduction byIL-1α or IL-1β. Another agent suitable for treating such diseases is asoluble CD30 polypeptide that binds CD30L, such as a polypeptidecomprising amino acids 19-390 of SEQ ID NO:6 or a CD30L-binding fragmentthereof.

[0012] In yet another aspect of the invention, provided also is ananimal model for screening therapeutic agents, the animal model beingcharacterized by carrying genetic modifications that inactivate its p55and p75 TNFα receptor proteins, and also by being geneticallysusceptible to experimentally-induced arthritis. In a preferredembodiment, the animal model is genetically susceptible iscollagen-induced arthritis. For example, inactivation of the p55 and p75TNFα proteins can be introduced into wild-type DBA/1, BUB or B10.Q miceor into DA, BB-DR or LEW rats, all of which are susceptible tocollagen-induced arthritis. In a preferred embodiment, the animal is aDBA/1 mouse that has been genetically modified so that it hasdouble-null mutations in both its p55 and p75 TNFα receptor genes.

[0013] The invention also provides methods for using the aforedescribedanimal model to screen a candidate therapeutic agent to determine itsefficacy in treating an autoimmune or chronic inflammatory conditionthat is resistant to treatment with a TNFα inhibitor. This methodcomprises assays in which one induces arthritis in an animal in whichthe p55 and p75 TNF receptors have been inactivated, administers thecandidate therapeutic agent to the animal, and determines that the agentis efficacious if the severity of the animal's arthritis is reducedafter the candidate agent has been administered. In a preferredembodiment, the screening assays employ a strain of mouse or rat that issusceptible to collagen-induced arthritis and in which the p55 and p75proteins have been inactivated by genetic modification. In such mice,arthritis is induced by injecting collagen, the candidate therapeuticagent is administered, and then the severity of the arthritis isassessed by observing the amount of erythema and edema in the animalspaws. In a preferred embodiment, the screening assays employ a DBA/1mouse, a BUB mouse, a B10.Q mouse, a DA rat, a BB-DR rat or a LEW rat. Aparticularly suitable animal for use in these assays is a DBA/1 mousecarrying double-null mutations in its p55 and p75 TNFα receptor genes.

DETAILED DESCRIPTION OF THE INVENTION

[0014] The instant invention discloses methods and compounds fortreating or preventing autoimmune and chronic inflammatory diseases,including conditions resistant to treatment with TNFα inhibitors, aswell as a model system for screening agents for their efficacy intreating autoimmune or inflammatory conditions that are refractory totreatment with TNFα inhibitors. Patients treated in accord with theinvention include those whose condition is continuous or intermittent.Diseases treatable by the subject methods include, for example, diseasessuch as arthritis, systemic lupus erythematosus and degenerativeconditions of the nervous system, such as multiple sclerosis.

[0015] Preferably, the patient undergoing treatment is a mammal, andmore preferably is a human. The subject methods are applicable todomestic animals, including pets and farm animals. Provided also hereinare methods for using inhibitors that block CD30 or IL-1 signaltransduction to treat autoimmune or chronic inflammatory diseases thatare resistant to treatment with inhibitors of TNFα. In addition, methodsare provided that involve treating the autoimmune or chronicinflammatory diseases described herein concurrently with two or moreinhibitors selected from an IL-4 inhibitor, a TNFα inhibitor and aninhibitor of the CD30/CD30L interaction. For purposes of thisdisclosure, the terms “illness,” “disease,” “medical condition,”“abnormal condition,” “malady,” “medical disorder”, “disorder” and thelike are used interchangeably.

[0016] Autoimmune conditions are characterized by the production ofantibodies or effector T cells that react with native host molecules.Most B cell responses depend on helper T cells, thus the presence ofautoantibodies generally involves some dysregulation of T cell function.In some cases, however, autoantibodies arise from normal T and B cellresponses to foreign proteins that share antigenic epitopes with thehost's own tissues. For example, autoantibodies may be elicited by apathogenic bacteria that expresses an antigen that resembles a hostmolecule. In some instances, arthritic syndromes may originate in awoman due to her exposure to fetal cells that have escaped into hercirculation during pregnancy. The phrase “chronic inflammatorycondition” as used herein refers to chronic disorders whose ongoingsymptoms do not appear to be caused by a viral or bacterial infection,even though these diseases-in some instances may have been triggered byan infection that no longer is present. Such diseases are “inflammatory”in that they are characterized by the release of inflammatory cytokinessuch as tumor necrosis factor (TNFα), lymphotoxin α and/or interleukin-1(IL-1), and they may also involve autoimmunity. In some cases, geneticpredispositions play a role in autoimmune or chronic inflammatorydiseases that are treatable by the subject methods. For such patients,the subject therapies may be administered prophylactically if desired.

[0017] In one aspect of the invention, autoimmune and chronicinflammatory diseases are treated by administering an agent thatinhibits signal transduction by CD30. An agent's ability to inhibit CD30signal transduction can be demonstrated using a biological assay, suchas an assay that involves determining that the agent interferes with abiological activity manifested by CD30⁺ cells that otherwise would occurwhen the cells are contacted with CD30L. Alternatively, a CD30antagonist may be identified by determining its ability to prevent CD30Lfrom binding CD30 that is expressed on the surface of cultured cells.Therapeutic agents of the invention include but are not limited toagents that antagonize the specific binding of CD30 to CD30L. The terms“antagonist” and “inhibitor” are used interchangeably herein.

[0018] The term “CD30-ligand” (CD30L) refers to a human or murineCD30-binding protein as disclosed in Smith et al. (1993) and U.S. Pat.No. 5,480,981, including CD30-binding muteins thereof. Nucleotide andamino acid sequences for human CD30L are shown in SEQ ID NOS:1 and 2,and those for mouse CD30L are shown in SEQ ID NOS:3 and 4. Theextracellular region of human CD30L corresponds to amino acids 1-215 ofSEQ ID NO:2. For forming soluble human CD30L molecules that can bindCD30, polypeptides comprising amino acids 44, 45, 46 or 47 through aminoacid 215 of SEQ ID NO:2 may be used. The extracellular region of murineCD30L corresponds to amino acids 1-220 of SEQ ID NO:4. For formingsoluble murine CD30L molecules that can bind CD30, polypeptidescomprising amino acids 49-220 of SEQ ID NO:4 may be used.

[0019] As used herein, the phrase “fragment of CD30L” refers to aportion of a full-length CD30L polypeptide that retains the ability tobind to CD30, or that is capable of eliciting an antibody that bindsspecifically with a CD30L polypeptide of SEQ ID NO:2 or 4 or asubportion thereof. Such fragments preferably will contain at least aportion of the extracellular region of CD30L.

[0020] The term “CD30” as used herein refers to the 595 amino acid humanCD30 polypeptide encoded by the nucleotide sequence of SEQ ID NO:5, andwhose amino acid sequence is shown in SEQ ID NO:6. The cloning of CD30is described in Durkop et al. (Cell 68:421 (1992)). The extracellularportion of human CD30 corresponds to amino acids 1-390 of SEQ ID NO:6,or if the signal peptide is removed, to amino acids 19-390 of SEQ IDNO:6. The phrase “soluble CD30” (sCD30) refers to soluble molecules thatcomprise all or part of the extracellular domain of the CD30 protein,and that retain the capacity to bind specifically with CD30L. SolubleCD30 polypeptides of the invention encompass recombinant sCD30 andnaturally-occurring sCD30 proteins in highly purified form. If desired,the sCD30 may be linked to polyethylene glycol (i.e., pegylated) toprolong its half-life in the patient's body, or may be linked to anotherprotein moiety that promotes oligomerization.

[0021] As used herein, the phrase “fragment of CD30” refers to a portionof a full-length CD30 polypeptide that retains the ability to bind toCD30L, or that is capable of eliciting an antibody that bindsspecifically with a CD30 polypeptide having the amino acid sequence SEQID NO:6 or subportion thereof or to a portion of full-length CD30 thatis capable of transmitting a biological signal such as activation ofNF-κB.

[0022] Soluble CD30 polypeptides according to the invention also includepolypeptides that are at least 60%, or at least 70%, or at least 80%,and most preferably at least 90% of the length of the extracellularregion of the human CD30 molecule. as shown in amino acids 1-390 of SEQID NO:6. Further included as therapeutic agents are proteins comprisinga soluble CD30 polypeptide having at least 80%, at least 85%, at least90%, at least 95%, at least 97.5%, or at least 99%, and most preferablyat least 99.5% sequence identity with amino acids 1-390 of SEQ ID NO:6,where sequence identity is determined by comparing the amino acidsequences of the two polypeptides when aligned so as to maximize overlapand identity while minimizing sequence gaps. The value for percentidentity in such comparisons can be determined by visual inspection andmathematical calculation. Alternatively, the percent identity of twoamino acid sequences can be determined by comparing sequence informationusing the GAP computer program, version 6.0 described by Devereux et al.(Nucl. Acids Res. 12:387, 1984) and available from the University ofWisconsin Genetics Computer Group (UWGCG). The preferred defaultparameters to be used for the GAP program in making these comparisonsinclude: (1) a unary comparison matrix (containing a value of 1 foridentities and 0 for non-identities) for nucleotides, and the weightedcomparison matrix of Gribskov and Burgess, Nucl. Acids Res. 14:6745,1986, as described by Schwartz and Dayhoff, eds., Atlas of PolypeptideSequence and Structure, National Biomedical Research Foundation, pp.353-358, 1979; (2) a penalty of 3.0 for each gap and an additional 0.10penalty for each symbol in each gap; and (3) no penalty for end gaps.Other programs used by those skilled in the art of sequence comparisonmay also be used, such as, for example, the BLASTN program version2.0.9, available for use via the National Library of Medicine website,or the UW-BLAST 2.0 algorithm, using standard default parameter settingsdescribed at the blast-wustl website. In addition, the BLAST algorithmuses the BLOSUM62 amino acid scoring matrix, and optional parametersthat may be used are as follows: (A) inclusion of a filter to masksegments of the query sequence that have low compositional complexity(as determined by the SEG program of Wootton & Federhen (Computers andChemistry, 1993); also see Wootton J C and Federhen S, 1996, Analysis ofcompositionally biased regions in sequence databases, Methods Enzymol.266: 554-71) or segments consisting of short-periodicity internalrepeats (as determined by the XNU program of Claverie & States(Computers and Chemistry, 1993)), and (B) a statistical significancethreshold for reporting matches against database sequences, or E-score(the expected probability of matches being found merely by chance,according to the stochastic model of Karlin and Altschul (1990); if thestatistical significance ascribed to a match is greater than thisE-score threshold, the match will not be reported.); preferred E-scorethreshold values are 0.5, or in order of increasing preference, 0.25,0.1, 0.05, 0.01, 0.001, 0.0001, 1e-5, 1e-10, 1e-15, 1e-20, 1e-25, 1e-30,1e-40, 1e-50, 1e-75, or 1e-100.

[0023] The phrase “CD30/CD30L interaction” as used herein refers to thespecific binding of CD30 to CD30L, resulting in signal transduction byCD30. This includes instances in which at least one binding partner is afragment of either CD30 or CD30L, that is, the term may refer to thebinding interaction of a CD30 fragment to CD30L, CD30 to a CD30Lfragment, or a CD30 fragment to a CD30L fragment. In addition, aCD30/CD30L interaction can involve an analog of CD30 (such as an allelicvariant or mutein) that is capable of binding specifically to CD30L, ormay involve an analog of CD30L (such as an allelic variant or mutein)that can bind specifically with CD30. Moreover, a CD30/CD30L interactioncan involve either endogenous CD30 or CD30L proteins or may involverecombinant CD30 or CD30L expressed by a cell transfected with a nucleicacid encoding the recombinant protein. Similarly, the phrase “IL-1/IL-1Rinteraction” refers to the specific binding between IL-1 and IL-1R,including instances where at least one of the binding partners is afragment of the full-length polypeptide, and including instances whereone of the binding partners is an allelic variant or mutein that retainsthe ability to bind specifically with its binding partner.

[0024] The term “IL-1” as used herein includes both IL-1α and IL-1β.IL-1α and IL-1β are two distinct proteins that both are instrumental ininflammation, and both of which bind the same two cell surfacereceptors. The cell surface receptors to which IL-1α and IL-1β both bindare known as types I and II IL-1 receptors, or “IL-1R1” and “IL-1R2.”Signal transduction by IL-1 is mediated primarily by IL-1R1, whileIL-1R2 is considered to be a “decoy” receptor whose function is todownregulate IL-1 biological activity. Human IL-1R2 is described in U.S.Pat. No. 5,350,683. A nucleic acid sequence encoding the IL-1R2 proteinis shown in SEQ ID NO:7, and the amino acid sequence is shown in SEQ IDNO:8.

[0025] In one embodiment, the subject invention provides methods oftreating autoimmune or chronic inflammatory conditions that areresistant to treatment with drugs that inhibit TNFα. In such patients,the lack of response to TNFα inhibition may be partial or complete. TNFαinhibitors to which these diseases are unresponsive may include one ormore of the following: etanercept (p75 TNFR:Fc, sold as ENBREL®; ImmunexCorporation); LENERCEPT® (p55 TNFR-Ig fusion protein; Roche); orantibodies against TNFα, including humanized antibodies such asinfliximab (REMICADE®; Centocor), D2E7 (BASF Pharma) or CDP571(HUMICADE®; Celltech). Arthritis that is resistant to one of these or toanother TNF inhibitor is referred to as “TNFα-independent arthritis.”Diseases resistant to treatment with TNFα inhibitors may be treated byadministering one or more agents that inhibit signal transduction byCD30 or IL-1, such as an inhibitor of the CD30/CD30L interaction and/orof the IL-1/IL-1R interaction. In addition, diseases that are less thanfully responsive to TNFα inhibitors are treated with a combination of aTNFα inhibitor administered concurrently with an inhibitor of theCD30/CD30L interaction and/or with an inhibitor of an IL-1/IL-1Rinteraction.

[0026] Therapeutic Agents

[0027] Any physiologically acceptable agent capable of blocking signaltransduction by CD30 may be used as a therapeutic agent in the disclosedmethods of treatment, including but not limited to: antibodies that bindspecifically to CD30L and thereby inhibit its binding to CD30;non-agonistic antibodies that bind specifically to CD30 and inhibit itsability to transmit a biological signal; muteins or analogs such asallelic variants of CD30L or fragments thereof that bind to CD30 butthat do not stimulate transduction of a biological signal; sCD30molecules that contain the extracellular domain of CD30 or a portionthereof that is capable of binding CD30L; and small organic moleculesthat block signal transduction by CD30 or that interfere with thebiological functioning of CD30L.

[0028] Soluble CD30 molecules used as therapeutic agents as describedherein comprise the extracellular region of CD30. An exemplary CD30protein is shwon in SEQ ID NO:6, in which amino acids 1-390 of SEQ IDNO:6 correspond to the extracellular region. If less than the entireextracellular domain is used, the fragment must retain the ability tobind CD30L, which ability may be ascertained using any convenientbinding assay format. The presence of the signal peptide in the sCD30 isoptional. Suitable assays include testing for the ability of the sCD30to competitively block binding of labeled CD30L to cells expressingsurface CD30 or to block binding of isolated CD30L or cells expressingsurface CD30L to cell-surface CD30 or CD30 that is anchored to a solidsupport such as an ELISA plate or a chromatography matrix, such asagarose beads. When used therapeutically, these sCD30s block the bindingof membrane-bound CD30 to cells expressing CD30L

[0029] sCD30s useful as CD30 antagonists for the disclosed therapeuticmethods include oligomeric sCD30 polypeptides (such as dimers ortrimers), as well as monomers. Oligomers may be linked by disulfidebonds formed between cysteine residues on different sCD30 polypeptides.In one embodiment of the invention, the therapeutic agent is a sCD30that is created by fusing the extracellular domain of CD30 (or a CD30Lbinding portion thereof) to the Fc region of an antibody, preferably ahuman antibody, in a manner that does not interfere with binding of theCD30 moiety to CD30L. The resulting fusion protein is a CD30:Fc thatwill dimerize by the spontaneous formation of disulfide bonds betweenthe Fc moieties on two of the fusion protein chains. Native Fc regionpolypeptides or muteins thereof may be employed in making theseconstructs. Suitable CD30:Fc's will reduce or abolish the ability ofagonistic anti-CD30 antibodies to stimulate CD30, or will competitivelyinhibit the binding of CD30L to membrane-bound CD30. An example of asuitable CD30:Fc protein that may be used in the subject methods is onethat is constructed as described in U.S. Pat. No. 5,677,430.

[0030] Other suitable oligomeric sCD30 polypeptides may be prepared byfusing, the extracellular domain of CD30 (or a fragment thereof) to a“leucine zipper,” which is a peptide that promotes oligomerization ofthe proteins in which they are present. Leucine zippers are motifs foundin several DNA-binding proteins (see, for example, Landschulz et al.,Science 240:1759 (1988)), and naturally-occurring leucine zipperpeptides and derivatives thereof can promote the formation of dimers ortrimers of protein chains in which they are present. Examples of leucinezipper domains useful for producing soluble oligomeric proteins aredescribed in WO 94/10308.

[0031] Another method of linking multiple copies of soluble CD30polypeptides is by fusing monomers together with suitable peptidelinkers. A fusion protein comprising two or more copies of sCD30separated by peptide linkers may be produced by recombinant DNAtechnology. Such peptide linkers optimally are from 5 to 100 amino acidsin length, preferably comprising amino acids selected from the groupconsisting of glycine, asparagine, serine, threonine, and alanine. Theproduction of recombinant fusion proteins comprising peptide linkers isillustrated in U.S. Pat. No. 5,073,627.

[0032] In yet other embodiments, antagonists can be designed to reducethe level of endogenous CD30 or CD30L gene expression, for example, byusing well-known antisense or ribozyme approaches to inhibit or preventtranslation of CD30 or CD30L mRNAs; triple helix approaches to inhibittranscription of CD30 or CD30L genes; or targeted homologousrecombination to inactivate or “knock out” the CD30 or CD30L genes ortheir endogenous promoters or enhancer elements. Such antisense,ribozyme, and triple helix antagonists may be designed to reduce orinhibit either unimpaired, or if appropriate, mutant CD30 or CD30L geneactivity. Techniques for the production and use of such molecules arewell known to those of skill in the art.

[0033] Antisense RNA and DNA molecules can act to directly block thetranslation of mRNA by hybridizing to targeted endogenous mRNA therebypreventing translation. Alternatively, antisense RNA or DNA can inhibitor prevent transcription of the target gene. The antisense approachinvolves designing oligonucleotides (either DNA or RNA) that arecomplementary to a CD30 or CD30L mRNA, or complementary to a portion ofthe target gene, such as a regulatory element that controlstranscription of the gene. Antisense nucleic acids should be at leastsix nucleotides in length, and are preferably oligonucleotides rangingfrom 6 to about 50 nucleotides in length.

[0034] In one embodiment of the invention, ribozyme molecules designedto catalytically cleave CD30 or CD30L mRNA transcripts are used toprevent translation of CD30 or CD30L mRNA and expression of CD30 orCD30L polypeptides. (See, e.g., WO 90/11364, or U.S. Pat. No.5,824,519).

[0035] Provided herein are therapies for treating autoimmune and chronicinflammatory conditions that are resistant to treatment with aninhibitor of TNFα. Therapies according to this embodiment of theinvention are administered to a patient who has already been observed tobe partially or fully unresponsive to treatment with a TNFα inhibitor.To treat a patient whose autoimmune or chronic inflammatory condition isresistant to a TNFα inhibitor, the patient is administered an effectiveamount of an agent that is capable of blocking the binding of CD30 toCD30L, such as one of the inhibitors described above. Alternatively, thepatient is administered an IL-1 antagonist that is capable of inhibitingsignal transduction by IL-1, such as by reducing the amount of IL-1produced in the patient's body, or by interfering with the binding ofIL-1 to IL-1R1. In the case of patients who are partially responsive totreatment with a TNFα inhibitor, treatments according to this embodimentof the invention may be administered concomitantly with a TNFαinhibitor. The therapeutic agents of this embodiment of the inventiongenerally are administered in the form of a physiologically acceptablecomposition.

[0036] In a further embodiment of the invention, patients are treatedwith an inhibitor of the CD30/CD30L interaction administeredconcurrently with an antagonist of IL-4. IL-4 is a cytokine with a broadrange of activities, and is expressed by CD30⁺ T cells. The cell surfacereceptors to which IL-4 binds are referred to as the “IL-4 receptor” or“IL-4R.” (see for example, U.S. Pat. No. 5,599,905). The interactionbetween IL-4 and its receptor can be inhibited by administering asoluble IL-4 receptor (sIL-4R), such as the sIL-4Rs described in U.S.Pat. No. 5,599,905. Preventing this interaction will hinder or preventIL-4-mediated biological activities. IL-4 can induce a chronicinflammatory effect in some diseases and can exacerbate some autoimmunedisorders. In such diseases, the infiltration and proliferation ofT_(H)2 cells is fueled by IL-4 and CD30 signal transduction. These cellscause the overproduction of IL-4. Accordingly, diseases where thisoccurs, including atopic dermatitis, asthma, systemic lupuserythematosus, scleroderma or pemphigus vulgaris, are effectivelytreated by administering an agent that inhibits IL-4 concurrently withan agent that inhibits the interaction between CD30 and CD30L. Thelatter preferably is an antibody that is specific for CD30L.

[0037] Methods of using IL-4 inhibitors to treat immune or inflammatoryresponses are illustrated, for example, in U.S. Pat. No. 5,767,065. IL-4antagonists that may be administered in combination with inhibitors ofthe CD30/CD3-L interaction include, but are not limited to, IL-4receptors (IL-4R) and other IL-4-binding molecules, IL-4 muteins andantibodies that bind specifically with IL-4 or IL-4 receptors therebyblocking signal transduction, as well as antisense oligonucleotides andribozymes targeted to IL-4 or IL-4R. Polyclonal or monoclonal antibodiesspecific for IL-4 or IL-4 receptor may be prepared using standardprocedures. Among the IL-4 receptors suitable for use as describedherein are soluble fragments of human IL-4R that retain the ability tobind IL-4. Such fragments retain all or part of the IL-4R extracellularregion and are capable of binding IL-4. In a preferred embodiment of theinvention, the patient is treated concurrently with sIL-4R and anantibody specifically for CD30L.

[0038] IL-4 receptors are described in U.S. Pat. No. 5,599,905; Idzerdaet al., J. Exp. Med. 171:861-873, March 1990 (human IL-4R); and Mosleyet al., Cell 59:335-348, 1989 (murine IL-4R), each of which is herebyincorporated by reference in its entirety. The protein described inthose three references is sometimes referred to in the scientificliterature as EL-4Rα. Unless otherwise specified, the terms “EL-4R” and“IL-4 receptor” as used herein encompass this protein in various formsthat are capable of functioning as IL-4 antagonists, including but notlimited to soluble fragments, fusion proteins, oligomers, and variantsthat are capable of binding IL-4. Suitable IL-4Rs include variants inwhich valine replaces isoleucine at position 50 (see Idzerda et al.,1990), and include slow-release formulations, and PEGylated derivatives(modified with polyethylene glycol) are contemplated, as well asrecombinant fusion proteins comprising heterologous polypeptides fusedto the N-terminus or C-terminus of an IL-4R polypeptide, includingsignal peptides, immunoglobulin Fc regions, poly-His tags or the FLAG®polypeptide described in Hopp et al., Bio/Technology 6:1204, 1988, andU.S. Pat. No. 5,011,912, as well as fusions of IL-4 receptors witholigomer-promoting leucine zipper moieties. Soluble recombinant fusionproteins comprising an IL-4R and immunoglobulin constant regions aredescribed, for example, in EP 464,533. A nucleotide sequence encodinghuman IL-4 receptor is shown in SEQ ID NO:15 and amino acid sequence forhuman IL-4 receptor is shown in SEQ ID NO:16. In a preferred embodiment,the IL-4 antagonist to be used in combination with an inhibitor of theCD30/CD30L interaction is a soluble human IL-4 receptor comprising aminoacids 1 to 207 of SEQ ID NO: 16 and in another preferred embodiment, theIL-4 antagonist comprises amino acids 2 to 207 of SEQ ID NO: 16. IL-4antagonists useful for the hereindescribed methods of treatment alsoinclude molecules that selectively block the synthesis of endogenousIL-4 or IL-4R, such as antisense nucleic acids or ribozymes targetedagainst either of these molecules.

[0039] Various IL-4 antagonists that may be used for the hereindescribedmethods of treatment can be identified, for example, by their ability toinhibit ³H-thymidine incorporation in cells that normally proliferate inresponse to IL-4, or by their ability to inhibit binding of IL-4 tocells that express IL-4R. In one assay for detecting IL-4 antagonists,one measures the ability of a putative antagonist to block theIL-4-induced enhancement of the expression of CD23 on the surfaces ofhuman B cells. For example, B cells isolated from human peripheral bloodare incubated in microtiter wells in the presence of IL-4 and theputative antagonist. Following the incubation, washed cells are thenincubated with labelled monoclonal antibody against CD23 (available fromPharmingen) to determine the level of CD23 expression. An anti-huIL-4Rmurine mAb (R&D Systems) previously shown to block the binding andfunction of both hIL-4 and hIL-13 may used as a positive control forneutralization of CD23 induction by IL-4. Alternatively, suitable IL-4antagonists may be identified by determining their ability to prevent orreduce the impaired the barrier function of epithelium that results whenIL-4 is incubated with the epithelium. For this purpose, one may useconfluent monolayers of human epithelial cell lines such as Calu-3(lung) or T84 (intestinal epithelium). Incubation of such monolayerswith IL-4 causes significant damage to their barrier function withinabout 48 hours. To assay IL-4 antagonists, monolayers may be tested fortheir permeability, for example, by adding radiolabeled mannitol tocells incubated with IL-4 in the presence or absence of an antagonist.Alternatively, transepithelial resistance (indicating an intact barrier)may be determined using a voltmeter.

[0040] In other embodiments of the invention, antagonists of theCD30/CD30L interaction are administered concurrently with an IL-1antagonist. Alternatively, IL-1 antagonists may be administered alonefor treating autoimmune or inflammatory conditions that are resistant totreatment with TNFα0 inhibitors. Suitable agents for inhibiting signaltransduction by IL-1 include antibodies specific for IL-1 or IL-1R1,particularly humanized antibodies. Other suitable IL-1 antagonistsinclude: IL-1 receptor antagonist (IL-1ra); receptor-binding fragmentsof IL-1 that block native IL-1 from binding IL-1R1; antibodies directedagainst IL-1 or IL-1R1; and recombinant proteins comprising all or partof a receptor for IL-1 or modified variants thereof, includinggenetically-modified muteins, multimeric forms and sustained-releaseformulations. IL-1ra is a naturally-occurring endogenous antagonist ofIL-1 and binds both IL-1R1 and IL-1Rs. Preferred IL-1 antagonistsinclude soluble IL-1R2 molecules that are capable of binding IL-1 andthat comprise all or part of the extracellular domain of IL-1R2. Thesesoluble IL-1R2 molecules block IL-1 from interacting with membrane-boundIL-1R1. Other useful IL-1 antagonists include soluble forms of IL-1R1that are capable of competitively inhibiting the interaction of IL-1with IL-1R1 and further include IL-1β converting enzyme (ICE)inhibitors, which generally are small organic molecules. A preferredIL-1 antagonist is a soluble fragment of IL-1R2 that includes aminoacids 1-333 of SEQ ID NO:8, or a subportion thereof that is capable ofbinding specifically with IL-1α or IL-1β. Soluble IL-1R2s to be used inaccord with the invention include, for example, analogs or fragments ofnative IL-1R2 having at least 20 amino acids, that lack thetransmembrane region of the native molecule, and that are capable ofbinding IL-1. The ability of soluble IL-1R2 to bind IL-1 (includingbinding to fragments of IL-1α or IL-1β) can be assayed using ELISA orany other convenient assay.

[0041] Other suitable IL-1 antagonists are -chimeric proteins in whichone of the IL-1 antagonists described above is fused with anotherpolypeptide that promotes the spontaneous formation of a dimer, trimeror higher order multimer. A suitable polypeptide moiety for promotingdimerization is the Fc region of a human immunoglobulin. For example,soluble IL-1R2 polypeptides or fragments thereof may be fused the Fcregion of an immunoglobulin to form a chimeric protein that is capableof dimerizing. Any of the foregoing IL-1 antagonists, other than ICEinhibitors, may be covalently linked to polyethylene glycol (pegylated)to prolong their half-life in the body.

[0042] It is understood that while IL-1α and IL-1β are the IL-1s mostcommonly associated with inflammation, other forms of IL-1 exist, andthe invention encompasses the use of therapeutic agents targeting theseother forms for treating autoimmune and chronic inflammatory diseasesthat are resistant to treatment with TNFα inhibitors.

[0043] Preferred agents for use in the subject therapeutic methodsinclude antibodies that block the CD30/CD30L, IL-1/IL-1R or IL-4/IL-4Rinteractions. Such antibodies are specifically immunoreactive with theirtarget, that is, they bind to the target protein via the antigen-bindingsite of the antibodies and do not bind unrelated proteins to asignificant degree. Antibodies specific for CD30L will bind endogenousCD30L, thus reducing the amount of endogenous CD30L available forbinding to cell surface CD30. Also suitable for use as a therapeuticagent of the subject invention are biologically active fragments ofantibodies. For example, a biologically active fragment of an anti-CD30Lantibody is an antibody protein that is truncated relative to the intactantibody, but that retains the ability to specifically bind CD30L and toblock its interaction with CD30. Antigen-binding fragments ofantibodies, include, but are not limited to, Fab and F(ab′)₂ fragments,and may be produced by conventional procedures.

[0044] Antibodies that antagonize the CD30, IL-1 or IL-4 signaltransduction can be identified in any suitable assay, including assaysbased on biological function or assays based on the detection ofphysical binding. Examples of such assays are disclosed, for example, inU.S. Pat. No. 5,677,430. One preferred assay tests an antibody's abilityto block the binding of cell surface CD30L to cell surface CD30. Celllines suitable for such assays include the CD30+ HDLM-2 or L540 celllines, or may use activated T cells expressing CD30. An assay based onbiological function, for example, could entail determining whether anantibody can antagonize the ability of membrane-bound CD30L to stimulateproliferation of CD30⁺ cells that are responsive to such stimulation.Yet another assay employs the CD30⁺ K299 human cell line. It has beenobserved that proliferation of these cells is inhibited by contact withCD30L-transfected cells. An antibody specific for CD30L (or otherantagonist that blocks CD30L) could suppress this effect of CD30L andthus enhance the proliferation of these cells in this assay. In otherinstances, the antibody is tested for its ability to block the bindingof labeled recombinant CD30L to cell surface CD30. In other instances,the assays employ cells transfected with DNA encoding human CD30L,IL-1R1, IL-1R2 or IL-4R. For example, the ability of a monoclonalantibody against human CD30L to block CD30/CD30L interactions can beassessed by determining whether it can block the binding of humanCD30:Fc to either cells transfected with human CD30L or to activatedhuman T cell blasts as assessed by FACS analysis. Similarly, thespecificity of an antibody for IL-4R could be tested by determining ifthe antibody blocks binding of labeled IL-4 to IL-4R.

[0045] Other suitable assays utilize soluble forms of both bindingpartners, for example, sCD30 and sCD30L. For example, sCD30 may be boundto a solid phase, such as a column chromatography matrix or to an ELISAplate. For an ELISA assay using sCD30 and sCD30L, a sCD30, such asCD30:Fc, is fixed to an ELISA plate, and an antibody raised againstCD30L is tested to see if it is an antagonist by checking its ability toinhibit the binding of a soluble CD30L leucine zipper construct to theanchored sCD30. In this assay, binding of the leucine zipper constructor other soluble CD30L to the ELISA plate is measured using abiotinylated non-neutralizing anti-CD30L monoclonal antibody, oralternatively, by using an antibody to the leucine zipper end of theCD30L construct. Other assays test the ability of an antibody to blockthe binding of IL-1 to cell surface IL-1R1.

[0046] Therapeutic agents suitable for use in the subject methodsinclude non-agonistic antibodies against human CD30. Such antibodies canblock the CD30/CD30L interaction. Monoclonal antibodies against humanCD30 can be prepared, for example, as described in U.S. Pat. No.5,677,430, then tested to determine if they are agonistic ornon-agonistic. A non-agonistic antibody against CD30 may bedistinguished from an agonistic antibody by testing its effect on CD30in a suitable biological assay, such as the assays described U.S. Pat.No. 5,677,430. In one such assay, an antibody specific for CD30 istested to determine whether it can induce proliferation of activated Tcells prepared from peripheral blood or whether it can induceproliferation of the Hodgkin's disease-derived cell lines HDLM-2 orL-540. An agonistic antibody, will induce such proliferation. Incontrast, a non-agonistic antibody against CD30 will bind specificallywith CD30 but will not induce proliferation of target cells in these orother assays that rely on signal transduction by CD30.

[0047] Therapeutic agents according to this invention may beadministered concurrently with one or more additional therapeuticmolecules to treat autoimmune or chronic inflammatory diseases. As usedherein, “concurrently” includes instances where the drugs in acombination treatment are administered over the same time period or arealternated. This includes simultaneous and sequential administration,and the different drugs may be present in the same or separatepharmaceutical compositions. The frequency and route of administrationfor the different drugs in such combinations may be the same ordifferent. Therapeutic agents that may be used in such combinationsinclude, for example, antagonists of CD30, IL-1 or IL-4 as describedabove, and also include non-steroidal anti-inflammatory drugs (NSAIDs),corticosteroids, analgesics, cytokine suppressive drugs,disease-modifying anti-rheumatic drugs (DMARDs), methotrexate, and soon. As an example, antagonists of CD30 or IL-1 may be combined with eachother or with antagonists of TNFα, IL-2, IL-6, RANK or other cytokinesthat may contribute to autoimmunity or chronic inflammation. In somepreferred embodiments, the additional therapeutic agents target acytokine. An antagonist that targets a cytokine may comprise a solublereceptor against the cytokine, and usually includes part or all of theextracellular domain of a receptor for the cytokine. The solublecytokine receptor may be used as a monomer, or as a dimer or highermultimer (for example, as a fusion molecule wherein the soluble receptoris attached to the dimer-promoting Fc portion of human immunoglobulin).In other embodiments, the soluble cytokine receptor is pegylated toincrease its serum half-life. In some embodiments, the soluble cytokineantagonist comprises a soluble TNF receptor (type I or II). Smallorganic molecules that inhibit inflammatory cytokines may also be usedin combination with the subject therapeutic agents. More than oneantagonist of CD30 signal transduction may be administered concurrentlyfor treating autoimmune or chronic inflammatory diseases, and may beadministered alone or together with other drugs that are effectiveagainst the same autoimmune or chronic inflammatory condition or thatare being administered to treat a different condition in the samepatient.

[0048] Combinations used to treat multiple sclerosis include inhibitorsof CD30 administered in conjunction with other drugs used to treat thiscondition, including but not limited to mitoxantrone (NOVANTRONE®;Immunex Corporation), interferon β-1a (AVONEX®; Ares-Sorono Group),interferon β-1b (BETASERON®; Berlex Laboratories, Inc.) and/orglatiramer acetate (COPAXONE®; Teva Pharmaceuticals). IL-4 antagonistsmay be added to any of the foregoing combination.

[0049] For treating various rheumatic conditions, including rheumatoidarthritis, an agent capable of inhibiting CD30 signal transduction maybe administered alone or concurrently with inhibitors of TNFα, such asantibodies against TNFα (for example, humanized antibodies such asREMICADE® (Centocor), D2E7 (BASF Pharma), or HUMICADE® (Celltech));soluble forms of the TNF receptor (such as ENBREL® (Immunex Corporation)or LENERCEPT® (Roche) or pegylated soluble TNF receptors.

[0050] Preparation of Therapeutic Antibodies

[0051] CD30L polypeptides suitable for use as an immunogen in producingtherapeutic anti-CD30L antibodies include but are not limited to fulllength CD30L (recombinant or prepared from a naturally-occurring source)or immunogenic fragments thereof, particularly fragments containing allor part of the extracellular domain of CD30L. To be effective as animmunogen, a fragment of CD30L need not retain the capacity to bindCD30, but must be large enough to be antigenic. To be antigenic, apeptide generally must contain at least 20 amino acids. The amino acidsequence of full-length human CD30L is shown in SEQ ID NO:2 and mouseCD30L in SEQ ID NO:4; peptides corresponding to at least 20 contiguousamino acids of either of these proteins may be used as an immunogen toprepare therapeutic agents for use as described herein.

[0052] CD30 polypeptides suitable for use as immunogens in producingnon-agonistic anti-CD30 antibodies include but are not limited to fulllength CD30 proteins (recombinant or prepared from a naturally-occurringsource) or immunogenic fragments thereof, particularly fragmentscomprising all or part of the extracellular domain as defined by aminoacids 1-390 of SEQ ID NO:6. Immunogens for raising anti-CD30 antibodiespreferably contain at least 20 contiguous amino acids of the proteinshown in SEQ ID NO:6.

[0053] IL-1 or IL-1R polypeptides suitable for use as immunogens inproducing antagonistic antibodies include but are not limited to fulllength proteins (recombinant or prepared from a naturally-occurringsource) or immunogenic fragments thereof, particularly fragmentscomprising all or part of the extracellular domain of human IL-1R2 asshown in amino acids 1-333 of SEQ ID NO:8. Preferred immunogens containat least 20 contiguous amino acids of the protein shown in SEQ ID NO:8.

[0054] Immunogens for raising therapeutic antibodies against TNFα, TNFR,IL-4 or IL4-R will consist of an at least 20 amino acid segment of thetarget protein. Suitable antigens for raising therapeutic antibodiesalso include any of the aforementioned immunogens fused to anotherprotein, including proteins fused to an N-terminal “flag” (see, forexample, Hopp et al., Bio/Technology 6:1204 (1988); U.S. Pat. No.5,011,912), or fused to the Fc portion of an immunoglobulin molecule,preferably a human immunoglobulin. Preferred therapeutic agents includeboth polyclonal and monoclonal antibodies (MABs), either of which may begenerated using the above-described polypeptides as immunogens.

[0055] Polyclonal antibodies may be generated according to standardprotocols using a variety of warm-blooded animals such as horses, cows,rabbits, mice, rats, or various species of fowl. In general, the animalis immunized with CD30L, CD30, IL-1, IL-1R1, TNFα, TNFR1 or TNFR2, or animmunogen derived therefrom, through intraperitoneal, intramuscular, orsubcutaneous injections. The immunogenicity of an immunogenicpolypeptide usually is increased through the co-administration of anadjuvant such as RIBI® (Corixa), or Freund's complete or incompleteadjuvant, or other suitable adjuvant. After several boosterimmunizations, serum samples are collected and tested for specificreactivity with the target polypeptide by any suitable method, such asELISA, antibody-capture (“ABC”) or modified ABC assays, or a dot blotassay. Once the titer of antibody has reached a plateau in terms ofreactivity to the target, the polyclonal antiserum is harvested.

[0056] For an ABC assay, a plastic dish, such as an ELISA plate, iscoated with an antibody that is specifically reactive with the Fcportion of immunoglobulin from for the same species of animal that wasused to raise the antibody against the target polypeptide. For example,if a target protein is was injected into a rabbit and the resultinganti-target polyclonal antibody is to be evaluated for specificity, theplates are coated with antibody specifically reactive with the Fcportion of rabbit IgG. In the next step, a sample of antibody from theimmunized rabbit is incubated in the dish under conditions that promotebinding between the rabbit IgG and the anchored anti-rabbit antibody.Next, labeled target protein is added, and the dish is incubated underconditions that promote antibody binding. If the sample of antibodybeing tested is specific for the target protein, then the labeled targetprotein will become bound to the captured rabbit antibody, andthereafter can be detected after the plate is washed. For example, ifthe target protein is labeled with biotin, target protein that hasbecome bound can be detected by using a streptavidin-tagged enzyme thatis capable of generating a colored product.

[0057] Suitable procedures for generating monoclonal antibodies includeseveral methods described in the art (see, for example, U.S. Pat. No.4,411,993; Kennett et al. (eds.), Monoclonal Antibodies, Hybridomas: ANew Dimension in Biological Analyses, Plenum Press, New York (1980); andHarlow and Lane (eds.), Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. (1988)). Mice or ratsgenerally are used for the initial immunizations, which are performed asdescribed above for raising polyclonals. After immunization, theimmunized animal's spleen cells are harvested and fused according tostandard procedures with a myeloma cell line to yield immortalizedhybridoma cells that produce monoclonal antibodies. Many myeloma linessuitable for hybridomas are known, including many that are availablefrom the American Type Culture Collection (ATCC), Rockville, Maryland(see Catalog of Cell Lines & Hybridomas, ATCC). Individual hybridomacells are isolated after the fusion step to be screened to identifythose producing monoclonal antibodies having the desired specificimmunoreactivity. For therapeutic use, high affinity antibodies arepreferred. Hybridomas with the desired specification are identified byassays such as ELISA, ABC, modified ABC, or dot blot assays, then areisolated and propagated. The monoclonal antibodies are harvested andpurified using standard methods.

[0058] Monoclonal antibodies against CD30L may be raised and screenedfor specific reactivity according to the methods described in U.S. Pat.No. 5,677,430 or by any of the various methods known in the art forraising monoclonals. Screening may involve determining the capacity ofthe monoclonals to antagonize the binding of cell-surface CD30L tocell-surface CD30 or to antagonize signal transduction by cell-surfaceCD30.

[0059] Antibodies useful for the subject therapeutic methods alsoinclude chimeric antibodies and antibodies produced or modified viaprotein engineering or recombinant DNA technology (see, for example,Alting-Mees et al., Strategies in Molecular Biology 3:1 (January,1990)). Production of chimeric and otherwise engineered antibodies isdescribed in the prior art (see, for example, Reichmann et al., Nature332:323 (1988); Liu et al., PNAS 84:3439 (1987); Larrick et al.,Bio/Technology 7:934 (1989); and Winter and Harris, TIPS 14:139 (1993)).

[0060] When an antibody is to be administered as a therapeutic agent toa human patient in accord with the subject methods, a humanized antibodyis preferred. Even more preferred are human antibodies. Humanizedantibodies comprise an antigen binding domain derived from a non-humanantibody (for example, a rat or mouse), and may contain the entirevariable region of the non-human antibody, or may contain only thatportion of the non-human variable region that includes theantigen-binding site. In a preferred embodiment of the invention, theonly non-human portion of the humanized antibody is the hypervariableregion. Procedures for preparing humanized antibodies are known, andinclude techniques that involve recombinant DNA technology. Productionof humanized antibodies is described, for example, in Winter and Harris,1993. To create a humanized antibody, DNA encoding the antigen-bindingsites of a monoclonal antibody directed against the target may beisolated and inserted directly into the genome of a cell that isproducing human antibodies (see Reichmann et al. (1988)). For example,this method could be used for antibodies against CD30, CD30L, IL-1,IL-1R1, TNFα, TNFR-1 or TNRF-2, IL-4 or IL-4R.

[0061] In one procedure for preparing humanized antibodies, cDNA isprepared on a mRNA template derived from a hybridoma cell line thatproduces a non-human monoclonal antibody having the desired specificity.In essence, a fragment of the cDNA that encodes the variable region ofthe monoclonal antibody (or a fragment thereof containing the antigenbinding site) is isolated, and this cDNA fragment is fused to DNAencoding the human counterpart of the remainder of the human antibodymolecule, thereby reconstructing a functional antibody molecule. Hostcells are transfected with an expression vector containing the fusedgene, and are cultured to produce the desired recombinant fusionprotein. To isolate DNA encoding the variable region of animmunoglobulin chain, one may employ, for example, the method of Larricket al. (1989), which involves the polymerase chain reaction (PCR), usinga mixture of upstream primers corresponding to the leader sequence, anda downstream primer based on the conserved sequence of the constantregion. PCR primers for amplifying the variable region from mouse orhuman hybridoma cell immunoglobulin mRNA are commercially available (forexample, from Stratacyte, La Jolla, Calif.). PCR primers may be used toamplify heavy or light chain variable region DNA, and the resultingamplified DNA may be inserted into vectors such as ImmunoZAP* H orImmunoZAP* L (Stratacyte), respectively, for expression in E. coli.

[0062] To produce a humanized antibody for use as a therapeutic agent inaccord with the invention, variable region DNA is isolated from a murineor rat hybridoma cell that expresses an antibody with the desiredspecificity, and this DNA is fused with a constant region DNA amplifiedfrom a cDNA encoding a human antibody. These or similar techniques maybe used, for example, to produce a single-chain antigen-binding proteincontaining a V_(L) domain fused to a V_(H) domain through a peptidelinker (see Bird et al., Science 242:423 (1988)). Further geneticmanipulations may be performed to replace all but the hypervariableregions of the antibody with human sequences.

[0063] Human antibodies may be generated using methods involvingnon-human animals. For example, DNA encoding one or more entire humanimmunoglobulin chains may be introduced into a mouse to produce atransgenic animal, and then the antibody is isolated from cultured cellsderived from the mice. The endogenous immunoglobulin genes in therecipient mouse may be inactivated by various means, and humanimmunoglobulin genes introduced into the mouse to replace theinactivated genes. These genetic manipulations will result in humanimmunoglobulin polypeptide chains replacing endogenous immunoglobulinchains in at least some instances, and in such mice some or most of theantibodies produced upon immunization will be human antibodies. Examplesof techniques for the production and use of such transgenic animals aredescribed in U.S. Pat. Nos. 5,814,318, 5,569,825, and 5,545,806.

[0064] Animal Model for Diseases Resistant to Treatment with TNFαInhibitors

[0065] TNF inhibition has demonstrated beneficial effects in asignificant percentage of rheumatoid arthritis patients and patientswith other kinds of arthritis or other chronic inflammatory diseases.However, a subset of rheumatoid arthritis patients respond poorly or notat all to treatment with TNF inhibitors (TNF-independent rheumatoidarthritis). To provide a tool for identifying effective treatments forpatients with autoimmune or chronic inflammatory conditions that showlittle or no improvement in response to treatment with TNFα inhibitors,provided herein is an animal model that is useful for screeningcandidate therapeutic agents that may be effective for such diseases.The model animal is characterized by carrying genetic modifications thatinactivate its p55 and p75 TNFα receptor proteins and by beinggenetically susceptible to experimentally-induced arthritis. The animalmodel is created by genetically modifying a strain of animal thatalready is known to be genetically susceptible to experimentally-inducedarthritis. Provided also are methods for screening candidate therapeuticagents to determine their effectiveness in treating a medical disorderthat is resistant to treatment with a TNFα inhibitor. Such medicaldisorders include rheumatoid arthritis and other kinds of arthritis.

[0066] The subject animal model is created by introducing geneticmodifications into a strain of animal, usually a mammal, that prior tomodification is genetically susceptible to experimentally-inducedarthritis. The genetic modifications result in the inactivation of theanimal's p55 and p75 TNFα receptor (TNFR) proteins. The p55 and p75TNFRs are also called the types I and II TNFRs, respectively.

[0067] A model animal according to the subject invention may bedeficient in its p55 and p75 TNFR proteins as the result of any ofseveral different types of mutation strategies. For example, thedeficiency may result from mutations that inhibit the transcription of atranslatable TNFR messenger RNA or that result in production ofdefective TNFR proteins that do not bind TNFα. Cells from the subjectanimal model will bind no detectable TNFα as compared with animals thatexpress at least one of these TNFR proteins in wild-type form. Theability of cells from a subject animal model to bind TNFα may beassayed, for example, as described in Peschon et al. (1998) or by othersuitable methods. For example, cells taken from a genetically modifiedanimal may be tested for expression of functional TNFR I and II by beingincubated with labeled biotinylated TNFα and streptavidin-conjugatedphycoerythrin, then analyzed by flow cytometry to determine if thephycoerythrin was captured on the cells. Various cells may be isolatedfrom the test animal to be used in such a binding assay, includingconconavalin A-stimulated thymocytes, thioglycolate-elicited peritonealexudate cells and bronchoalveolar lavage cells collected afterintranasal administration of lipopolysaccharide (Peschon et al., 1998).If such cells from the mutated animal fail to bind labeled TNFα, thisindicates that both types I and II TNFR are suitably inactivated.

[0068] In a preferred embodiment of the invention, the animal in whichthe p55 and p75 proteins are inactivated is a rodent strain that issusceptible to experimental collagen-induced arthritis (CIA). In apreferred embodiment, mutations are introduced into this rodent's genomethat result in inactivation of the genes encoding the p55 and p75 TNFαreceptors. In one preferred embodiment, the rodent is a strain of mouseor rat. Mice susceptible to CIA include mice that carry the H-2^(q) MHChaplotype or the H-2^(r) MHC haplotype. Exemplary strains ofCIA-susceptible mice include the DBA/1, BUB and B10.Q strains, andexemplary strains of CIA-susceptible rats include the DA, BB-DR and LEWstrains (see, for example, Joe and Wilder, Mol Med Today 5:367-369(1999) and Anthony and Haqqi, Clin Exp Rheumatol 17:240-244 (1999)).

[0069] An exemplary animal model according to the invention is a DBA/imouse carrying double-null mutations in its p55 and p75 TNFR genes, thatis, a p55^(−/−)p75^(−/−) DBA/1 mouse. As used herein, a “null” mutationmeans that the gene is sufficiently changed relative to the wild-typegene such that it does not give rise to a protein that is recognizableby antibodies specific for the corresponding wild-type receptor protein.This may be accomplished by introducing a deletion or insertion into thewild-type gene, or by other means. Once established in a strain ofmouse, a null mutation may be transferred to a different strain byappropriate genetic manipulations. A double-null mutation means thatboth alleles of that gene carry a null mutation.

[0070] In other embodiments, the genetic modifications may be introducedinto rodent strains that are susceptible to forms of arthritis otherthan CIA, such as, for example, one of the arthritis-susceptible strainsthat are described in Joe and Wilder (1999).

[0071] Provided herein are methods that employ the subject animal modelfor screening assays to determine whether a candidate therapeutic agentis effective for treating autoimmune or chronic inflammatory conditionsthat are resistant to treatment with a TNFα inhibitor. TNFα inhibitorsto which these diseases are unresponsive may include receptor-based TNFαinhibitors such as ENBREL® (Immunex Corporation) and LENERCEPT® (Roche)or other drugs that incorporate a soluble TNFR, humanized antibodiesagainst TNFα such as REMICADE® (Centocor), D2E7 (BASF Pharma) or CDP57 1(HUMICADE®; Celltech) or small molecules whose pharmacologiceffectiveness may be based on reducing endogenous TNFα (such as, forexample, pentoxifylline, thalidomide or others).

[0072] Candidate therapeutic agents that are tested in the subjectassays are determined to be effective if when administered to thesubject animal model the agent brings about a reduction in the severityof arthritis that has been induced in the animal. The severity ofarthritis in the animal may be assessed by any desired method, which maybe based, for example, on assigning to each animal a numerical scorethat reflects the degree of swelling or stiffness of the animal's limbs.Efficacy of a test agent in reducing the severity of disease isdetermined by comparing this score averaged over a group of arthriticanimals that receive the test agent with the score for a group thatreceives a placebo. The test agent and placebo generally areadministered over a period of at least one week, but may be administeredfor a longer period, for example, over a period of 2, 3, 4, 5, 6, 7 or 8or more weeks. Alternatively, the effects of a single dose may beassessed using this model.

[0073] If DBA/1 p55^(−/−)p75^(−/−) mice with CIA are used in the assays,an efficacious therapeutic agent will partially or fully ameliorate thesymptoms of CIA in the mice. A reduction in severity of arthritis isdetermined to be present in DBA/1 p55^(−/−)p75^(−/−) mice if the averageclinical score (determined as described below) for an agent-treatedgroup of mice is one or more clinical score units lower than the averageclinical score for a group of negative control mice that are given aplacebo. Preferably, the average clinical score in the agent-treatedDBA/1 p55^(−/−)p75^(−/−) mice will be at least 2 clinical score unitslower than the placebo-treated mice, and more preferably it will be atleast 5 units lower.

[0074] To determine clinical score for individual DBA/1p55^(−/−)p75^(−/−) mice in which CIA has been induced, the followingindex may be used in which each paw is assigned a score in clinicalunits based on the following:

[0075] 0=normal appearance

[0076] 1=erythema/edema in 1-2 digits

[0077] 2=erythema/edema in >2 digits, or mild swelling in ankle/wristjoint

[0078] 3=erythema/edema in entire paw

[0079] 4=massive erythema/edema of entire paw extending into proximaljoints;

[0080] ankylosis, loss of function

[0081] Paw scores are combined for each mouse to determine a final scorefor that mouse, and then final scores for all animals in the test agentgroup are averaged and compared with the average score for theplacebo-treated group. Generally, each test group will contain between 5and 30 animals, though smaller or larger numbers of animals may be used.This or similar clinical scoring systems are suitable for evaluating CIAin DBA/1 p55^(−/−)p75^(−/−) mice, but also are suitable for otherspecies and for animal models involving types of experimental arthritisother than CIA. If desired, other kinds of arthritis scoring systems maybe used, such as, for example, a system based on levels in the blood orother tissues of molecules that reflect the degree of inflammation ormolecules that are specific disease markers.

[0082] When DBA/1 p55^(−/−)p75^(−/−) mice are used, the CIA is inducedaccording to the procedure given in Example 2, or using a similarprotocol. In one preferred screening assay, a test agent is administeredto a DBA/1 p55^(−/−)p75^(−/−) mouse that is subjected to CIA induction,with the first dose of the agent being administered on the day thecollagen boost is given, or the first dose may be delayed until theonset of symptoms, which generally take 15-60 days to appear in thesemice. In this assay, negative control DBA/1 p55^(−/−)p75^(−/−) mice aretreated with a placebo, such as rat or mouse IgG or a physiologicallyacceptable saline solution. If desired, an agent known to be efficaciousagainst CIA in these mice can be included in the assay as a positivecontrol; for example, antibody against CD30L or against II-1R1 can beused as a positive control (see Example 4), although this is optional.Doses of the test agent and control agent(s) may be administered daily,every two days, every three days, two times per week, one time per week,or less often if desired. The duration of the testing period isvariable, and may continue, for example, for three days, one week, twoweeks, three or for four or more weeks.

[0083] In the subject assays, the test agent and placebo may beadministered by any suitable route, including orally administered liquidor solid forms, topical application, aerosol inhalation, transfection ofhost cells by recombinant DNA expressing the test agent, or byinjection, including intraperitoneal, intravenous, subcutaneous orintramuscular injection. If desired, the test agent may be administeredvia a slow-release implant. When the test agent is incorporated into aslow-release formulation, very few doses are required, and a single dosemay be used. If the test agent is an antibody and the subject is amouse, the candidate therapeutic test agent may be administered at adose of 20-75 μg/mouse, and is administered every day, every two to fourdays, or once a week. If the test agent is a small molecule, such as anorganic molecule, suitable doses for testing are from 0.5-1000 μg/mouse,and the agent may be administered once every one to ten days. If alarger species of model animal is used, dose is adjusted upward inproportion to average body weight for the larger species.

[0084] Efficacy of the test agent in DBA/1 p55^(−/−)/p75^(−/−) mice oranother strain of animal is determined by comparing the percentage ofanimals affected with arthritis in test and negative control groups, orby comparing the percentage of animals exhibiting severe disease duringthe testing period, and/or by comparing the mean clinical score forcontrol groups and test groups after administration of the test agent.Mean clinical score may be determined according to the index describedin Example 2, or by other suitable means. A test agent is determined tobe useful for treating disorders resistant to TNFα inhibitors if animalsto whom the test agent is administered exhibit a reduced severity ofdisease as compared with control animals that receive placebo instead ofthe test agent. In one preferred embodiment, the observed improvement isstatistically significant, that is, p<0.05. Optionally, a dosedependency of the therapeutic response is established by administeringthe test agent at several different doses.

[0085] Candidate therapeutic agents to be screened in the subject animalmodel include any agent that is potentially effective for treatingdiseases that are resistant to treatment with TNFα inhibitors. Thisincludes, for example, agents that target cytokines other than TNFα thatare sometimes associated with inflammation (such as, for example,GM-CSF, interferon-γ, lymphotoxin-α, IL-1, IL-4, IL-8, IL-15, IL-17 andIL-18). Candidate therapeutic agents include soluble receptor moleculesand antibodies specific for target cytokines or their receptors. Smallorganic molecules may also be screened using the subject animal model,including but not limited to agents with potential for interfering withCD30 or IL-I signal transduction

[0086] Therapeutic Methods

[0087] Disclosed herein are methods for treating a variety of autoimmuneand chronic inflammatory diseases by administering to a patient in needthereof an effective amount of one of the above-described agents.Blockers of CD30 signal transduction are used for treating any of themedical disorders listed below, and blockers of CD30 or IL-1 signaltransduction are used for treating disorders listed below that respondpoorly or not at all to treatment with a TNFα antagonist. In someinstances, a disease is generally responsive to treatment with a TNFαinhibitor, but unresponsive in certain patients. An example of such adisease is rheumatoid arthritis. Rheumatoid arthritis patients whorespond poorly or not at all to TNFα inhibitors will particularlybenefit from treatment with an antagonist of the CD30/CD30L orIL-1/IL-R1 interaction.

[0088] Preferably, the patient is a human, and may be either a child oran adult. In one embodiment of the invention, a condition believed to beT_(H)2 driven Or a condition characterized by high levels of expressionof CD30 on activated T cells, is treated by administering a CD30inhibitor concurrently with an IL-4 inhibitor.

[0089] For the subject therapeutic methods, the therapeutic agentspreferably are administered in the form of a physiologically acceptablecomposition comprising a purified recombinant protein in conjunctionwith physiologically acceptable carriers, excipients and/or diluents.Such carriers are nontoxic to recipients at the dosages andconcentrations employed. Compositions suitable for in vivoadministration may be formulated according to methods well-known in theart. Components that are commonly employed in such formulations includethose described in Remington's Pharmaceutical Sciences, 16th ed., 1980,Mack Publishing Company. Ordinarily, the preparation of suchcompositions entails combining the therapeutic agent with buffers,antioxidants such as ascorbic acid, low molecular weight polypeptides(such as those having fewer than 10 amino acids), proteins, amino acids,carbohydrates such as glucose, sucrose or dextrins, chelating agentssuch as EDTA, glutathione and other stabilizers and excipients. Neutralbuffered saline or saline mixed with non-specific serum albumin areexemplary appropriate diluents. If desired, the therapeutic agent may beformulated as a lyophilizate using appropriate excipient solutions suchas sucrose as a diluent. Appropriate dosages can be determined instandard dosing trials, and may vary according to the chosen route ofadministration. In accordance with appropriate industry standards,preservatives may also be added, such as benzyl alcohol.

[0090] The amount and frequency of administration may vary, depending onsuch factors as the nature and severity of the indication being treated,the desired response, the duration of treatment, the age, weight andcondition of the patient, and so forth. The dose of a therapeutic agentmay be adjusted to accommodate various routes of administration, oraccording to the needs of individual patients as determined by thepatient's physician.

[0091] Arthritis may be treated by the methods and compositionsdisclosed herein. As used here, the term “arthritis” refers to chronicinflammatory conditions that primarily affect joints, or the connectivetissue surrounding joints, although various body organs may also becomeaffected. Arthritis may be autoimmune or traumatic in origin, or it maybe triggered by exposure to a foreign antigen, thereafter leading to achronic condition that is no longer dependent on the continued presenceof the triggering antigen. The term “arthritis,” as used herein,includes: arthritis deformans; osteoarthritis; rheumatoid arthritis(adult and juvenile); Lyme disease arthritis; reactive arthritisincluding Reiter's disease; psoriatic arthritis; arthritis nodosa;seronegative spondylarthropathies, including but not limited toankylosing spondylitis. The efficacy of anti-CD30L treatment in treatingarthritic disease is illustrated in Examples 2 and 4.

[0092] The subject inhibitors, compositions and combinations are usefulin treating a variety of rheumatic disorders, which are defined hereinas any chronic disorder involving painful and often multiple localizedinflammations of the joints, muscles, nerves, tendons, skin, eyes,connective tissues or various other organ systems. These include but arenot limited to: arthritis; scleroderma; gout; systemic lupuserythematosus; polymyalgia rheumatica; fibromyalgia; Still's disease;chronic uveitis; disorders resulting in inflammation of the voluntarymuscle, including dermatomyositis and polymyositis, including sporadicinclusion body myositis; and inflammatory conditions such as chronicback or neck pain and sciatica. Systemic lupus erythematosus can causeinflammation of the joints, skin, kidneys, heart, lungs, blood vesselsand brain. In its advanced forms, systemic lupus erythematosus thiscondition can result in kidney failure. Treatment with antibody againstCD30L appeared to delay the progression of kidney failure in a mousemodel for this disease (see Example 6).

[0093] Provided also are methods for using the subject inhibitors,compositions or combination therapies to treat various disorders of theendocrine system, including but not limited to: juvenile or maturityonset diabetes (including autoimmune, insulin-dependent types ofdiabetes; non-insulin dependent types and obesity-mediated diabetes);idiopathic adrenal atrophy; Addison's disease; hypothyroidism; Grave'sdisease; autoimmune thyroiditis, such as Hashimoto's thyroiditis; andpolyglandular autoimmune syndromes (types I and II).

[0094] Conditions of the gastrointestinal system also are treatable withthe subject inhibitors, compositions or combination therapies, includingbut not limited to: autoimmune sclerosing cholangitis; coeliac disease;inflammatory bowel diseases, including Crohn's disease and ulcerativecolitis; autoimmune pancreatitis, including chronic pancreatitis;idiopathic gastroparesis; and idiopathic ulcers, including gastric andduodenal ulcers.

[0095] Included also are methods for using the subject inhibitors,compositions or combination therapies for treating disorders of thegenitourinary system, such as autoimmune and idiopathicglomerulonephritis; and chronic idiopathic prostatitis (non-bacterial),including benign prostatic hypertrophy.

[0096] Also provided herein are methods for using the subjectinhibitors, compositions or combination therapies to treat varioushematologic disorders, including but not limited to: anemias andhematologic disorders, including pernicious anemia and aplastic anemia,and Fanconi's aplastic anemia; autoimmune hemolytic anemia; idiopathicthrombocytopenic purpura (ITP); myelodysplastic syndromes (includingrefractory anemia, refractory anemia with ringed sideroblasts,refractory anemia with excess blasts, refractory anemia with excessblasts in transformation); and autoimmune lymphoproliferative syndrome(ALPS).

[0097] In addition, the subject inhibitors, compositions and combinationtherapies are used to treat hereditary conditions such as Gaucher'sdisease, Huntington's disease, and muscular dystrophy.

[0098] The disclosed inhibitors, compositions and combination therapiesare furthermore used to treat conditions that affect the liver such asautoimmune or chronic inflammatory hepatitis that is not due to viralinfection.

[0099] In addition, the disclosed inhibitors, compositions andcombination therapies are used to treat various autoimmune or chronicinflammatory disorders that involve hearing loss. One of these is innerear or cochlear nerve-associated hearing loss that is thought to resultfrom an autoimmune process, i.e., autoimmune hearing loss. Thiscondition currently is treated with steroids, methotrexate and/orcyclophosphamide, which may be administered concurrently with aninhibitor of the CD30/CD30L interaction.

[0100] A number of inflammatory pulmonary disorders also can be treatedwith the disclosed inhibitors, compositions and combination therapies,including: idiopathic lymphangioleiomyomatosis; chronic obstructivepulmonary disease (COPD) associated with chronic non-infectiousbronchitis or with emphysema; and fibrotic lung diseases, such as cysticfibrosis and idiopathic pulmonary fibrosis.

[0101] Disorders associated with transplantation also are treatable withthe disclosed inhibitors, compositions or combination therapies,including graft-versus-host disease. To prevent or ameliorategraft-versus-host disease, the subject inhibitors may be administeredprior to, concomitantly with, or following bone marrow or solid organtransplantation, including transplantation of heart, liver, lung, skin,kidney or other organs.

[0102] The subject inhibitors and the disclosed compositions andcombination therapies also are useful for treating chronic inflammatoryeye diseases, including autoimmune uveitis.

[0103] The subject inhibitors and the disclosed compositions andcombination therapies also are useful for treating inflammatorydisorders that affect the female reproductive system, including:multiple implant failure/infertility; fetal loss syndrome or IV embryoloss (spontaneous abortion); and endometriosis.

[0104] Other medical disorders treatable with the disclosed inhibitors,compositions and combination therapies include chronic degenerativediseases of the central nervous system. This includes, for example,diseases associated with demyelination, such as multiple sclerosis,systemic sclerosis and the Guillain-Barre syndromes (including acuteinflammatory demyelinating polyneuropathy, acute motor axonalneuropathy, acute motor sensory axonal neuropathy and Fisher syndrome).In a preferred embodiment, multiple sclerosis is treated with anantagonist of the CD30/CD30L interaction (most preferably an antibodyagainst CD30L) alone or concurrently with a TNFα inhibitor or an IL-4inhibitor (most preferably sIL-4R). Multiple sclerosis is representativeof a chronic, degenerative disease of the central nervous system, whichbesides the demyelinating conditions also include, for example,amyotrohpic lateral sclerosis (Lou Gehrig's Disease); Bell's palsy;Parkinson's disease and idiopathic chronic neuronal degeneration, all ofwhich may be treated with an agent capable of inhibiting the interactionof CD30 and CD30. The efficacy of anti-CD30L antibody in ameliorating amultiple sclerosis-like disease is illustrated in Example 5.

[0105] Other chronic inflammatory conditions treatable with thedisclosed inhibitors, compositions and combination therapies includecold agglutinin disease; Behcet's syndrome; Sjogren's syndrome; andidiopathic tenosynovitis, as well as various chronic inflammatorydisorders associated with hereditary deficiencies. The subjectinhibitors, compositions and combination therapies furthermore areuseful for treating Bell's palsy (idiopathic facial paralysis); chronicfatigue syndrome (not associated with ongoing infection); chronicdegenerative vertebral disc disease; Gulf war syndrome; and myastheniagravis, which may be treated concurrently with corticosteroids.

[0106] Disorders involving the skin or mucous membranes also aretreatable using the subject inhibitors, compositions or combinationtherapies. These include: acantholytic diseases, including Darier'sdisease, keratosis follicularis, pemphigus vulgaris and paraneoplasticpemphigus; acne rosacea; alopecia areata; bullous pemphigoid; eczema;erythema, including erythema multiforme and erythema multiforme bullosum(Stevens-Johnson syndrome); inflammatory skin disease; lichen planus;linear IgA bullous disease (chronic bullous dermatosis of childhood);loss of skin elasticity; neutrophilic dermatitis (Sweet's syndrome);pityriasis rubra pilaris; psoriasis; pyoderma gangrenosum; loss of skinelasticity; and toxic epidermal necrolysis.

[0107] Other diseases that can be treated with the disclosed compounds,compositions and combination therapies include: autoimmune-associatedchronic mucocutaneous candidiasis; allergies; sarcoidosis; multicentricreticulohistiocytosis; Wegener's granulomatosis; arteritis, includinggiant cell arteritis; vasculitis and chronic autoimmune myocarditis.

[0108] To treat a medical disorder using the compounds and compositionsprovided herein, a therapeutically effective amount of a therapeuticagent according to the invention is administered to a mammal in needthereof. The agent is administered according to a regimen of dose andfrequency of administration that is adequate to induce a sustainedimprovement in at least one indicator that reflects the severity of thedisorder. An improvement is considered “sustained” if the patientexhibits the improvement on at least two occasions separated by at leastone day, but preferably that are separated by one week, two weeks, threeweeks or four or more weeks. The severity of the disorder is determinedbased on signs or symptoms, or may be determined by questionnaires thatare administered to the patient, such as the quality-of-lifequestionnaires often used by physicians to assess the status of chronicdisease conditions.

[0109] One or more indicators that reflect the severity of a patient'sillness may be assessed for determining whether the frequency andduration of drug treatment is sufficient. The baseline value for achosen indicator is established by examination of the patient prior toadministration of the first dose of the therapeutic agent. Preferably,the baseline examination is done within about 60 days of administeringthe first dose.

[0110] For example, if the condition being treated is an arthriticcondition, such as rheumatoid arthritis, psoriatic arthritis,osteoarthritis, one or more indicators for determining sufficiency oftreatment may be chosen from among: number of tender, painful or swollenjoints; degree of joint pain or tenderness or swelling; patientself-assessment (e.g., quality-of-life questionnaires), and physicianassessment. For many arthritic diseases, the patient's self-assessmentis a satisfactory indicator. Typical self-assessment questionnaires willreflect a patient's ability to conduct their daily activities, theirperception of well-being, their level of pain and so on. The duration oftreatment required to induce a measurable improvement for an arthriticor any other type of disease treatable as described herein is typicallyone to several weeks.

[0111] If the condition being treated is multiple sclerosis, suitableindicators for determining sufficiency of treatment include observing animprovement in one or more of the following: bladder and bowel control;fatigue; spasticity; body or hand tremors; muscle weakness; ability towalk; numbness in limbs; ability to concentrate (e.g., performance on asimple memory test); and subjective level of pain. Alternatively, theindicator may consist of the patient's score on a quality of lifequestionnaire as described above.

[0112] If the condition being treated is systemic lupus erythematosus,the indicator for determining sufficiency of treatment may consist of anobserved improvement in one of the following: fatigue; fever; ulcers ofthe mouth and nose; facial rash (“butterfly rash”); photosensitivity(SLE often flares up after exposure to sunlight); pleuritis;pericarditis; Raynaud's phenomenon (reduced circulation to fingers andtoes with exposure to cold); kidney function; and white blood cell count(SLE patients often have decreased numbers of white blood cells).

[0113] Improvement in a patient's condition is induced by repeatedlyadministering a dose of a therapeutic agent according to the inventionuntil the patient manifests an improvement over baseline for the chosenindicator or indicators. In treating chronic conditions, a satisfactorydegree of improvement usually is obtained after repeatedly administeringthe agent or agents over a period of at least a month or more, e.g., forone, two, or three months or longer. In some instances, improvement mayoccur sooner than one month, for example, after three weeks, two weeks,one week, or even after a single dose. A treatment duration of one tosix weeks, or even a single dose, may be sufficient for treatingoccasional flare-ups in patients suffering from chronic conditions thattend to go into remission in-between flare-ups. For persistentconditions, treatment may be continued indefinitely if desired. Evanafter a condition has shown improvement, maintenance therapy may becontinued indefinitely at the same level or at a reduced dose orfrequency of administration. If the dose or frequency of administrationhas been reduced, it may be resumed at the previous level if thepatient's condition should worsen. In addition, treatment withinhibitors of the subject invention may be administered prophylacticallyto patients who are predisposed to an autoimmune or chronic inflammatorycondition.

[0114] Any efficacious route of administration may be used totherapeutically administer the subject therapeutic agents, combinationsand compositions. If injected, the agents can be administered, forexample, via intra-articular, intravenous, intramuscular, intralesional,intraperitoneal or subcutaneous routes. Bolus injection or continuousinfusion may be used. Other suitable means of administration includesustained release from microparticles, implants or the like, aerosolinhalation, eyedrops, oral preparations, including pills, syrups,lozenges or chewing gum, and topical preparations such as lotions, gels,sprays, ointments or other suitable techniques.

[0115] Alternatively, proteinaceous agents, such as antibodies orantigen-binding fragments thereof, may be administered by implantingcultured cells that express the protein. In one embodiment, thepatient's own cells are induced to produce the therapeutic agent bytransfection in vivo or ex vivo with a DNA that encodes a protein thatblocks the CD30/CD30L, IL-1/IL-1R, IL-4/IL-4R or TNF/TNFR interactions.This DNA can be introduced into the patient's cells, for example, byusing naked DNA or liposome-encapsulated DNA that encodes the agent, byusing calcium-phosphate precipitated DNA, or by other means oftransfection. Autologous cells that are transfected ex vivo are returnedto the patient's body.

[0116] Regardless of the route of administration that is chosen, it isunderstood that the treatment regimens may be adjusted depending on thepatient's needs, in accord with general principles of medicine.

[0117] When the therapeutic agent is an antibody, such as, for example,an antibody against CD30, CD30L, IL-1, IL-1R1, TNFα, IL-4 or IL-4R,preferred dose ranges for therapeutic or prophylactic purposes in humansinclude 0.1 to 20 mg/kg, or more preferably, 0.5-10 mg/kg. Anotherpreferred dose range is 0.75 to 7.5 mg/kg, as exemplified in theexperiments of Example 2 (adjusted for human body weight according toDeVita et al., eds., Cancer—Principles & Practice of Oncology, 4^(th)Ed., J. B. Lippincott, 1993). A larger or smaller amount of antibody perdose may be used to accommodate differences in affinity of the antibodyfor the antigen. A suitable dose range for CD30:Fc is 0.01-20 mg/kg ofbody weight, or more preferably, 0.1-10 mg/kg of body weight. For othersoluble proteins used as inhibitors in accord with the invention,suitable dose ranges include 2-500 μg/kg, 0.5-10 mg/kg and 10 to 50mg/kg of body weight. It is understood that the skilled physician willadjust the dose and frequency of administration in accord with the needsof the patient and the nature of the disease being treated.

[0118] The following examples are offered by way of illustration, andnot by way of limitation. Those skilled in the art will recognize thatvariations of the invention embodied in the examples can be made,especially in light of the teachings of the various references citedherein.

EXAMPLE 1 Monoclonal Antibodies Directed Against CD30L

[0119] A monoclonal antibody directed against murine CD30L was producedin rats as follows. Lewis rats were repeatedly immunized by theintraperitoneal route with 10-20×10⁶ transfected CHO cells expressingfull length murine CD30L. When serum titers were detected in the rats,they were given an intravenous boost with 10×10⁶ transfected CHO cells.After three days, spleen cells from the immunized rats were fused withAG8.653 mouse myeloma cells. When hybridomas were well established in96-well plates, supernatants from each well were screened by an ELISAassay that employed CHO cells that were transfected with DNA encodingCD30L (CELISA). For the CELISA, CHO cells expressing the recombinantCD30L were adhered to the ELISA plates, and each supernatant wasscreened for its ability to react with the CD30L expressed on the CHOcells. Hybridoma cells from positive wells were expanded in 48-wellplates and screened by CELISA and also by fluorescence-activated cellsorting (FACS) using transfected and non-transfected CHO cells.Hybridoma cells that bound transfected but not non-transfected CHO cellswere selected and screened further by FACS against mouse lymphoma cellsthat naturally express CD30L (EL4 cells). Hybridoma isolates thatrecognized both recombinant and naturally expressed CD30L were clonedtwice by limiting dilution cloning, during which the activity of thesupernatants was tracked at each step by CELISA and/or FACS assays. Oneof these clones, the M15 clone, was chosen for further propagation. Inadditional experiments, it was determined that the M15 antibodyspecifically blocked the binding interaction between murine CD30 andCD30L.

EXAMPLE 2 Treatment of Collagen-Induced Arthritis in Mice

[0120] Arthritis treatments that are effective in treatingcollagen-induced arthritis (CIA) are also effective in treatingarthritis in humans (see, for example, Anthony and Haqqi, Clin ExpRheumatol 17:240-244 (1999)), hence the effects of antagonizing theCD30/CD30L interaction was tested in CIA mice. Collagen-inducedarthritis (CIA) was elicited in male DBA/1 mice (Harlan, UK) byinjecting the mice with type II chicken collagen (Sigma). Mice wereinjected on day 0 with 100 μg of the collagen in complete Freund'sadjuvant, and boosted on day 21 with a dose of 200 μg in) incompleteFreund's adjuvant. Collagen injections were administered intradermallyat the base of the tail. In this model, generally 75-100% of the miceare affected, that is, 75-100% of the mice exhibit arthritis symptomsafter collagen injection. In these experiments, indicia of CIA usuallyappeared in affected mice by day 23.

[0121] Mice injected with collagen as described above were injectedintraperitoneally with doses ranging from 0.15-150 μg of the M15monoclonal antibody whose preparation is described in Example 1. Fourexperiments were conducted, each involving 13-15 mice per test group. Ineach of the four experiments, positive control mice received 150 μg ofetanercept (ENBREL®, Immunex Corporation), which is known to beeffective against CIA, and negative control mice received this sameamount of human IgG (huIgG) or rat IgG. Daily injections of M15,etanercept or IgG were initiated on day 21, i.e., the day of the secondcollagen injection, and were continued until day 33.

[0122] During these experiments, mice were assessed three times per weekfor clinical signs of arthritis by an independent observer blinded tothe treatment groups. Disease was evaluated using an arthritis indexsystem that has been established for this model system. For scoring,each paw was assigned a clinical score based on the index. Paw scoreswere combined for each animal to determine a clinical score for thatanimal. The index used was as follows:

[0123] 0=normal appearance

[0124] 1=erythema/edema in 1-2 digits

[0125] 2=erythema/edema in >2 digits, or mild swelling in ankle/wristjoint

[0126] 3=erythema/edema in entire paw

[0127] 4=massive erythema/edema of entire paw extending into proximaljoints;

[0128] ankylosis, loss of function

[0129] Improved mean clinical scores were evident in all groups thatreceived M15 by the fifth day of M15 administration, and in oneexperiment by the third day of administration. Final results of the fourexperiments are summarized in Tables 1-4, shown below. Mean clinicalscores, percent disease incidence (mice exhibiting CIA divided by totalnumber of mice in group), and percent of affected mice exhibiting severedisease were calculated for each group of mice. Mice considered to have“severe disease” are those that had a clinical score greater than two atany time during the experiment. Statistical significance was determinedfor the differences in mean clinical score between the negative controlgroup and the other groups of mice, based on scores on the last day ofthe experiment. Statistical significance was determined using a one-wayanalysis-of-variance (ANOVA) with Dunnett's method (H. J. Motuisky,Analyzing Data with GraphPad Prism, 1999, GraphPad Software, Inc., SanDiego, Calif.). One-way ANOVA compares three or more groups when thedata are categorized in one way. Dunnett's method compares controlgroups to treatment groups.

[0130] The first experiment tested the effects of administering to CIAmice a dose of 150 μg/day of the M15 antibody. By day 4 or 5 of thisexperiment, mean clinical score differences began to appear betweennegative control animals and those that received M15 or etanercept. Theresults of this first experiment are summarized in Table 1. As wasexpected, the mice that received etanercept (positive control group)exhibited a lower incidence of disease and a reduced incidence of severedisease when compared with the negative control mice that receivedHuIgG. The mice that received M15 also exhibited a lower diseaseincidence and a reduced percentage of mice with severe disease (seeTable 1) as compared with negative controls. In addition, the M15 grouphad a lower mean clinical score than the negative controls on the lastday of the experiment (day 33). For both the etanercept and the M15groups, the last day differences in mean clinical score relative tonegative controls were found to be statistically significant (p =0.05 orless). TABLE 1 % Disease Severe Mean Clinical Score on TreatmentIncidence Disease Last Day of Experiment M15 53% 27% 2.53 etanercept 33% 7% 0.53 HuIg 87% 80% 6.47

[0131] A second experiment employed a similar protocol to compare dosesof 50 μg and 150 μg of M15. The results are shown in Table 2. For thisexperiment, statistically significant improvement in mean clinical scorewas seen at the end of the experiment for both the etanercept group andfor both doses of M15. TABLE 2 % Disease Severe Mean Clinical Score onTreatment Incidence Disease Last Day of Experiment M15, 150 μg 40% 20%1.2 M15, 50 μg 33% 20% 1.07 etanercept 47%%  6% 0.73 HuIg 87% 80% 7.53

[0132] In a third experiment, doses of 15, 50 and 150 μg of M15 werecompared, and the results are summarized in Table 3. Again, mice thatreceived M15 experienced a lower incidence of disease, as well as alower incidence of severe disease as compared with negative controls. Astatistically significant improvement in mean clinical score vis-a-viscontrols was observed at all three doses of M15 in this experiment.TABLE 3 % Disease Severe Mean Clinical Score Treatment Incidence Diseaseon Last Day M15, 150 μg 42% 42% 3.29 M15, 50 μg 71% 57% 3.71 M15, 15 μg47% 47% 3.3 etanercept 53% 20% 1.33 HuIg 87% 80% 8.4

[0133] In a fourth experiment, 0.15, 1.5, 15 and 150 μg doses of M15were tested, using rat IgG as the negative control. Results of thisexperiment are shown in Table 4. As seen in Table 4, improvement in meanclinical score exhibited a dose-dependency at the higher doses. For the15 and 150 μg doses, the improvement in clinical score was statisticallysignificant. TABLE 4 % Disease Severe Mean Clinical Score TreatmentIncidence Disease on Last Day M15, 150 μg 33% 26% 1.47 M15, 15 μg 80%33% 2.67 M15, 1.5 μg 73% 67% 5.47 M15, 0.15 μg 67% 67% 5.67 etanercept40% 13% 1 rat IgG 87% 67% 5.8

EXAMPLE 3 TNF-Independent Model of Murine Collagen-Induced Arthritis

[0134] A novel mouse was generated by moving previously describedtargeted null mutations in both the p55 and the p75 TNFα receptors(Peschon et al., J Immunol 160:943-952, 1998) from the CIA-insensitiveC57BL-6 genetic background to the CIA-sensitive DBA/1 geneticbackground.

[0135] DBA/1 mice doubly deficient in p55 and p75 receptors (DBA/1p55^(−/−)p75^(−/−)) were generated by crossing C57BL/6p55^(−/−)p75^(−/−) mice (Peschon et al., 1998) with DBA/1 mice obtainedfrom Jackson Laboratories. The resulting double heterozygotes werecrossed again to DBA/1 mice. The resulting progeny that wereheterozygous for both mutations were further tested for homozygosity atthe DBA/1 MHC complex (required for CIA sensitivity) by FACS analysisusing antibodies specific for C57BL/6 and DBA/1 MHC alleles. Antibodiesagainst the C57BL/6 and DBA/1 MHC alleles were purchased from BDPharMingen.

[0136] Those progeny that were homozygous for the DBA/1 MHC were crossedto DBA/1 for another three generations to generate DBA/1 miceheterozygous for both p55 and p75 TNF receptor mutations (DBA/1 N5p55^(±)p₇₅ ^(±); “N5″ refers to the fifth backcross generation). DBA/1N5 p55^(±)p75^(±) mice were intercrossed to establish a colony of DBA/1mice doubly deficient in p55 and p75 TNF receptors (DBA/l N5p55^(−/−)p75^(−/−)). In order to identify mice that were homozygous foreach of the null mutations, DNA from the progeny of the latter crosseswas analyzed using PCR assays specific for the murine p55 and p75 TNFRgenes, using DNA extracted from ear punches.

[0137] For tracking mutations in the p55 gene, the following PCR primerswere used: p60-B: 5′-GGATTGTCAC GGTGCCGTTG AAG-3′ (SEQ ID NO:9) p60-E:5′-CCGGTGGATG TGGAATGTGT G-3′ (SEQ ID NO:10) p60-spe: 5′-TGCTGATGGGGATACATCCA TC-3′ (SEQ ID NO:11) pgk5′-66: 5′-CCGGTGGATGTGGAATGTGTG-3′(SEQ ID NO:12)

[0138] Twenty-five pmole each of the four primers listed above wereadded to the ear punch DNA and the mixture subjected to 32 cycles of PCRfor 1 minute at 94°, 1 min. 65° and 30 seconds at 72°. PCR products wereresolved and visualized on 3% USB fine resolution agarose (cat. #73422)gels run in TAE buffer and stained in ethidium bromide. Expected PCRproducts using the above primers were 120 bp for p55^(+/+), and 155 bpfor p55^(−/−). Heterozygous mice were expected to yield both products.Additional nonspecific products migrating at about 300-500 bp were seenoccasionally in p55^(−/−) mice.

[0139] The following primers were used to track the p75 mutations:p80-Kas: 5′-AGAGCTCCAGGCACAAGGGC-3′ (SEQ ID NO:13) p80i-1:5′-AACGGGCCAGACCTCGGGT-3′ (SEQ ID NO:14) pgk5′-66:5′-CCGGTGGATGTGGAATGTGTG-3′ (SEQ ID NO:12)

[0140] These PCR reactions used 50 pmole p80-Kas, 100 pmole p80i-1 and20 pmole pgk5′-66 for 32-34 cycles for 1 minute at 94°, 1 minute at 65°and 30 seconds at 72°. PCR products were resolved and visualized on 3%USB fine resolution agarose gels run in TAE buffer and stained inethidium bromide. p75⁺ ”+mice were expected to yield a 275 bp product,and p75^(−/−) mice to yield a 160 bp product. Heterozygotes wereexpected to yield both products. Additional nonspecific products wereoccasionally seen migrating at about 50-100 bp.

[0141] Homozygous DBA/1 p55^(−/−)p75^(−/−) mice were thus identifiedusing PCR and thereafter were interbred.

EXAMPLE 4 Collagen-Induced Arthritis in DBA/1 p55^(−/−)p75^(−/−) Mice

[0142] The following experiments using the p55^(−/−)p75^(−/−) DBA/1 miceof Example 3 demonstrate that TNFα-independent CIA can be effectivelytreated either by administering a test agent that inhibits signaltransduction by CD30 or IL-1.

[0143] CIA was induced in p55^(−/−)p75^(−/−) DBA/1 mice by administeringheterologous type II collagen according to the protocol described abovein Example 2. Two experiments were conducted, each using 15 mice pertest group. These experiments were designed to determine whether theinhibitors being tested could prevent the development of CIA in thesemice. Mice were randomly divided into groups (n=15) at the time of theboost and were injected daily with either the test agent or the controlprotein for 14 days.

[0144] Compared with wild-type DBA/1 mice, the DBA/1 p55^(−/−)p75^(−/−)mice injected with collagen displayed a delayed onset and slower courseof disease. However, significant clinical symptoms appeared in themutant mice 15-60 days after the second collagen injection. As expected,no diminution of arthritis symptoms was observed in DBA/1 p55^(−/31) p₇₅^(−/−) mice when they were treated with p75 TNFR.Fc (ENBREL®; ImmunexCorporation). In contrast, ENBREL® is highly effective in reducingarthritis symptoms in wild-type DBA/1 mice with CIA (see Example 2above). Thus, the arthritis observed in the p55^(−/−)p75^(−/−) DBA/1mice is not mediated by TNFα.

[0145] Experiments were conducted to determine whether molecules otherthan TNFα played a role in CIA in DBA/1 p55^(−/−)p75^(−/−) mice. Inthese experiments, agents that inhibit the CD30/CD30L or the IL-1IL-1R1interaction were tested to see if they would affect CIA in this animalmodel. Monoclonal antibodies against either IL-1R1 (M147; ImmunexCorporation) or murine CD30L (M15; preparation described in Example 1)were administered by intraperitoneal injection to these mice, whilecontrol mice received rat IgG. Each experimental group consisted of 15mice. Antibody treatment was initiated at day 21 (at the time of thecollagen boost), and was administered for 21 days for experiment #1, andfor 28 days in experiment #2. For M15 antibody, a dose of 50 μg wasadministered per day, and for the M147 antibody, a dose of 50 μg wasadministered every two days. Clinical score was determined three timesper week, using the clinical scoring system described in Example 2.

[0146] As illustrated in Table 5 (experiment #1) and Table 6 (experiment#2), the administration of M147 or M15 significantly reduced arthritisin the DBA/1 p55^(−/−)p75^(−/−) mice, with M147 resulting in an almostcomplete amelioration of disease. TABLE 5 % Disease Severe Mean ClinicalScore Treatment Incidence Disease on Last Day M15   26% 0.06% 0.75 M1470.06%   0% 0.07 rat IgG   67%   60% 4.27

[0147] TABLE 6 % Disease Severe Mean Clinical Score Treatment IncidenceDisease on Last Day M15 60% 40% 2.7 M147  0%  0% 0 rat IgG 80% 67% 4.1

[0148] The results of these two experiments show that collagen-inducedarthritis can be established in a TNFα-independent manner, and thatinhibiting either the IL-1/IL-1R1 or CD30/CD30L interactions effectivelyreduce disease in these mice.

EXAMPLE 5 Mouse Experimental Allergic Encephalomyelitis Model

[0149] This example demonstrates the efficacy of antagonists of theCD30/CD30L interaction for treating multiple sclerosis. A mouse modelfor multiple sclerosis was employed for this purpose. Chronicexperimental autoimmune encephalomyelitis (EAE), which is a wellaccepted experimental model for this disease, was induced in femaleC57BL/6 mice (Taconic Farms Inc., Germantown, N.Y.) using a modificationof the protocol described by Mendel et al. (Eur. J. Immunol. 25:1951-59,1995). In brief, disease induction involved the immunization of micewith the MOG35-55 peptide derived from rat myelin oligodendrocyteglycoprotein (Mendel et al., 1995). Modifications to the diseaseinduction protocol of Mendel et al. included the use of a lower dose ofMOG35-55 for immunization (see below), no booster immunization, and theuse of RIB® adjuvant instead of complete Freund's adjuvant.

[0150] To induce EAE, groups of age and weight-matched mice (11-13 miceper group) were given a dose of 100 μg of rat MOG35-55. The MOG35-55 wasemulsified in 0.2 ml RIBI adjuvant (Corixa Corporation), and injectedsubcutaneously at three sites distributed over the shaved flank. Toinduce EAE with accelerated onset, the mice in a second experiment(Experiment 2) received by intravenous injection 500 ng pertussis toxin(List Biological Laboratory Inc, Campbell, Calif.), which wasadministered 48 hours after they received their dose of MOG35-55. Themice in Experiment 1 received no pertussis toxin, thus disease onset inExperiment 2 was accelerated as compared with Experiment 1.

[0151] Administration of antibody or placebo was initiated on the dayafter the MOG35-55 was administered (day 1) and was continued throughday 11. Each mouse was injected intraperitoneally every other day with0.2 ml pyrogen-free phosphate-buffered saline (PBS) or 0.2 ml PBScontaining one of the following: (i) 100 μg M15 (anti-CD30L); (ii) 100μg rat IgG (Sigma); or (iii) 75 μg M147 (anti-IL1R1). Endotoxin levelswere <10 EU/mg of protein for all reagents. Mice were monitored dailyfor 35 days (Experiment 1) or 30 days (Experiment 2) for weight loss,disease onset and severity of clinical signs of EAE by an independentobserver blinded to the treatment groups.

[0152] The severity of EAE was assessed using a standard EAE indexsystem in which “0” is used to indicate an asymptomatic mouse andclinical scores ranging from 0.5 to 4 was used to indicate varyingdegrees of ascending paralysis. The severity of EAE was assessed using aslightly modified version of a commonly used EAE scoring system. In thissystem, “0” were used to indicate a mouse with no evidence of diseaseand scores of 1-5 were used to indicate varying degrees of ascendingparalysis as follows: 1, tail paralysis; 2, hind limb weakness; 3,partial hind limb paralysis; 4, complete hind limb paralysis; 5,moribund or dead. The disease protocol described above induces an acuteepisode of disease in control mice (peak score of 2-4) from which mostrecover at least partially. Thus the acute episode of disease is notlethal and mice do not reach a score of 5. The aforedescribed scale wasmodified to include a score of “0.5” which was given to mice that showedthe earliest signs of EAE but that did not exhibit complete paralysis ofthe tail. Mice given a score of 0.5 exhibited some or all of thefollowing symptoms: overnight weight loss of 1-2 grams; noticeabletremor when held up by the tail; and weakness at the distal tip of thetail.

[0153] The median day of onset of EAE was determined by Kaplan-MeierSurvival analysis. Significant differences in onset between groups wereassessed using a Log-Rank comparison. Fischer's exact test was used toanalyze the statistical significance of differences in the incidence ofEAE among the groups of mice.

[0154] Results of these two experiments demonstrated the amelioratingeffects of either of the tested antibodies on the onset, incidence andseverity of the clinical course of EAE. As shown below in Table 7,administration of either anti-CD30L (M15) or anti-IL-1R1 (M147) resultedin delayed disease onset and a reduced incidence of disease in both ofthe experiments. TABLE 7 Incidence (%) Combined Median Day of OnsetGroup Expt 1 Expt 2 Results Expt 1 Expt 2 PBS 10/11 11/13 21/24 (88%) 2216 (91%) (85%) rat IgG 9/11 12/13 21/24 (88%) 21 16 (82%) (92%) anti-6/11 8/13 14/24 (58%) 35 23 CD30L (55%) (62%) anti- 5/11 9/13 14/24(58%) not 25 IL-1R (45%) (69%) determined

[0155] In Table 7, for the combined results for mice that received M15or M147, when compared with the rat IgG group, p<0.05 for the incidenceof disease. In Experiment 1, for median day of onset for the M15 mice,p=0.067 vs Rat IgG and p<0.05 vs PBS. In Experiment 2 in Table 5, formedian day of onset, p<0.005 vs rat IgG for both the M15 and M147groups.

[0156] For these same two experiments, Table 8 shows the mean percentchange in body weights within each group over the acute course ofdisease. As shown in Table 8, mice that received either anti-CD30L oranti-IL-1R1 antibodies lost less weight during this time than mice thatreceived rat IgG or PBS. TABLE 8 Mean Percent Body Weight Mean PercentBody Change ± SEM Weight Change ± SEM Treatment (All mice) (Affectedmice only) Expt 1 anti-CD30L  −2.0 ± 2.4*  −6.2 ± 2.7 anti-IL-1RI  −1.5± 1.9**  −5.4 ± 3.2 PBS  −7.3 ± 3.1  −9.2 ± 2.7 Rat IgG  −9.7 ± 1.8−11.4 ± 1.75 Expt 2 anti-CD30L    0.7 ± 2.1***  −5.3 ± 2.0** anti-IL-1RI −7.7 ± 3.5* −13.3 ± 3.7 PBS −16.3 ± 2.9 −19.9 ± 1.8 Rat IgG −19.9 ± 2.9−21.7 ± 2.6

[0157] To calculate the weight data in Table 8, the baseline weight foreach mouse was defined as its weight on day 12 (Experiment 1) or day 10(Experiment 2) relative to immunization. These days were chosen asreference points because all of the mice in each experiment wereweight-matched on day 0 and the mean weight of all the groups increasedin a similar manner between days 0-12 (Experiment 1) or days 0-10(Experiment 2). The mean weights of the various groups diverged afterthese time points due to weight loss associated with the onset of EAE.The left-hand column of Table 8 shows the mean percent body weightchange calculated for all of the mice in each treatment group, that is,both the clinically affected (clinical score of at least 0.5) andnon-affected mice were included for this calculation. The right-handcolumn of Table 8 shows the mean body weight change during the acutephase of the disease for only the mice that were clinically affected.Body weight change for the affected mice was calculated based on thedifference between the baseline weight of each individual mouse and theminimum weight observed for that mouse after the onset of disease. Forthose mice that never showed clinical evidence of disease, the percentbody weight change for Table 8 was calculated by comparing the baselineweight of each non-affected mouse to its weight on day 25 of theexperiment.

[0158] The data in Table 8 illustrate that either anti-CD30L oranti-IL-R1 is effective in slowing the weight loss otherwise observed inmice injected with MOG35-55. The Student's t test was used to determinethe statistical significance of these body weight differences. Numbersfound to be statistically significant are marked with asterisks in Table8, and p values were as follows: *p<0.05 vs rat IgG controls; **p<0.01vs rat IgG controls; ***p<0.001 vs rat IgG controls.

[0159] Table 9 presents the clinical score results for Experiments 1 and2. Group means and SEM were calculated based on the peak clinical scorefor each mouse (0 for non-affected mice; 0.5 to 4 for affected mice).The mean peak clinical score for each antibody-treated group wascompared with the rat IgG-treated group using the Student's t test, andthe p values are shown in the last column of Table 9. TABLE 9 Mean PeakClinical Score Treatment (± SEM (scale 0-4)) p vs Rat IgG Expt 1anti-CD30L 1.1 ± 0.3 0.0621 anti-IL-1RI 0.9 ± 0.3* 0.0362 PBS 2.0 ± 0.30.7402 Rat IgG 2.1 ± 0.4 NA Expt 2 anti-CD30L 1.3 ± 0.4* 0.0122anti-IL-1RI 1.3 ± 0.3* 0.0056 PBS 2.2 ± 0.3 0.3262 Rat IgG 2.7 ± 0.3 NA

[0160] As shown in Table 9, significant differences in clinical scorewere observed between groups of mice treated with either M15 or M147, ascompared with the control mice. Differences that were determined to bestatistically significant (p<0.05) are marked in Table 9with anasterisk.

EXAMPLE 6 Blocking CD30L Delays Kidney Failure in a Murine Model ofSystemic Lupus Erythematosus

[0161] Female (NZB×NZW)_(F1) hybrid mice (referred to hereafter as“NZB/W mice”) spontaneously develop a lupus-like disease characterizedby the presence of serum autoantibodies to double-stranded DNA (dsDNA).These mice, which eventually experience total kidney failure, are oftenused as a model for experimentation directed at better understanding andtreating lupus in humans. Over time, this condition in NZB/W miceprogresses to kidney malfunction as manifested by the appearance ofproteinuria. In a trial experiment to study the onset and progression ofdisease in these mice, about half had serum anti-dsDNA titers and about10% had proteinurea by 26 weeks of age. By 38 weeks of age, 20% of themice had died, and of the remaining mice, 52% were proteinurea positiveand 98% had serum anti-dsDNA titers.

[0162] Female NZBJW mice 32 weeks of age were used in an experiment todetermine whether anti-CD30L could delay the development of lupus-likedisease. Within a group of 32 week old mice, 98% were serum positive forantibodies against dsDNA (detected by ELISA), but only 40% hadprogressed to renal disease as assessed by proteinuria (detected usingCHEMSTRIP®; Roche). Mice from this group that were negative forproteinuria were used in the following experiment.

[0163] Thirty-two week old NZ/B mice that did not have proteinuria weredivided into two groups, and each group was treated every other day with150 μg of either anti-CD30L (M15) (group of 10 mice) or rat IgG as acontrol (group of 9 mice), administered by intraperitoneal injection.Treatments were continued for five weeks, and the mice were assessedweekly for the presence of proteinuria. Approximate values for thepercentages calculated from the results of this experiment aresummarized in Table 10 below. TABLE 10 Percent Incidence Progression toProteinuria Treatment Week 1 Week 2 Week 3 Week 4 Week 5 Anti-CD30L 10%10% 10% 10% 20% Rat IgG  0% 10% 20% 30% 40%

[0164] The data in Table 10 shows a trend toward decreased incidence ofproteinuria in mice treated with anti-CD30L as compared with controlmice. A scale from 1-5 was used to assess the degree of proteinureaamong these mice. At five weeks, the mean proteinurea index for controlmice in this experiment was 1.7, whereas M15-treated mice had a meanproteinurea index of 0.6. This result suggests that humans sufferingfrom systemic lupus erythematosus might benefit from treatment withantibodies against CD30L or with other antagonists of the CD30/CD30Linteraction.

[0165] To confirm the result presented above, a second group of femaleNZB/W mice are randomly assigned to treatment groups as antibodiesagainst dsDNA first appear in their serum. This experiment is designedto test the effects of treatment on the progression of anti-dsDNA titersand the progression of proteinurea. One group of mice is treated with150 μg of anti-CD30L (Ml5), and the other group is treated with rat IgGas a control. Treatments are administered three times per week byintraperitoneal injection for a period of three weeks. Mice aremonitored weekly for titers of serum antibodies to dsDNA and for theappearance of proteinuria.

[0166] In other experiments, the efficacy of anti-CD30L treatment willbe tested in a model of chemically induced lupus. In this model,administration of the isoprenoid alkane pristane (2,6,10,14tetramethylpentadecane) induces autoantibody production and immunecomplex mediated glomerulonephritis. One advantage of this model is thatit is not restricted to a particular mouse strain. Initial experimentsare underway in normal BALB/c and C57BL/6 strains of mice.

1 16 1 705 DNA Homo sapiens CDS (1)..(705) 1 atg gac cca ggg ctg cag caagca ctc aac gga atg gcc cct cct gga 48 Met Asp Pro Gly Leu Gln Gln AlaLeu Asn Gly Met Ala Pro Pro Gly 1 5 10 15 gac aca gcc atg cat gtg ccggcg ggc tcc gtg gcc agc cac ctg ggg 96 Asp Thr Ala Met His Val Pro AlaGly Ser Val Ala Ser His Leu Gly 20 25 30 acc acg agc cgc agc tat ttc tatttg acc aca gcc act ctg gct ctg 144 Thr Thr Ser Arg Ser Tyr Phe Tyr LeuThr Thr Ala Thr Leu Ala Leu 35 40 45 tgc ctt gtc ttc acg gtg gcc act attatg gtg ttg gtc gtt cag agg 192 Cys Leu Val Phe Thr Val Ala Thr Ile MetVal Leu Val Val Gln Arg 50 55 60 acg gac tcc att ccc aac tca cct gac aacgtc ccc ctc aaa gga gga 240 Thr Asp Ser Ile Pro Asn Ser Pro Asp Asn ValPro Leu Lys Gly Gly 65 70 75 80 aat tgc tca gaa gac ctc tta tgt atc ctgaaa aga gct cca ttc aag 288 Asn Cys Ser Glu Asp Leu Leu Cys Ile Leu LysArg Ala Pro Phe Lys 85 90 95 aag tca tgg gcc tac ctc caa gtg gca aag catcta aac aaa acc aag 336 Lys Ser Trp Ala Tyr Leu Gln Val Ala Lys His LeuAsn Lys Thr Lys 100 105 110 ttg tct tgg aac aaa gat ggc att ctc cat ggagtc aga tat cag gat 384 Leu Ser Trp Asn Lys Asp Gly Ile Leu His Gly ValArg Tyr Gln Asp 115 120 125 ggg aat ctg gtg atc caa ttc cct ggt ttg tacttc atc att tgc caa 432 Gly Asn Leu Val Ile Gln Phe Pro Gly Leu Tyr PheIle Ile Cys Gln 130 135 140 ctg cag ttt ctt gta caa tgc cca aat aat tctgtc gat ctg aag ttg 480 Leu Gln Phe Leu Val Gln Cys Pro Asn Asn Ser ValAsp Leu Lys Leu 145 150 155 160 gag ctt ctc atc aac aag cat atc aaa aaacag gcc ctg gtg aca gtg 528 Glu Leu Leu Ile Asn Lys His Ile Lys Lys GlnAla Leu Val Thr Val 165 170 175 tgt gag tct gga atg caa acg aaa cac gtatac cag aat ctc tct caa 576 Cys Glu Ser Gly Met Gln Thr Lys His Val TyrGln Asn Leu Ser Gln 180 185 190 ttc ttg ctg gat tac ctg cag gtc aac accacc ata tca gtc aat gtg 624 Phe Leu Leu Asp Tyr Leu Gln Val Asn Thr ThrIle Ser Val Asn Val 195 200 205 gat aca ttc cag tac ata gat aca agc accttt cct ctt gag aat gtg 672 Asp Thr Phe Gln Tyr Ile Asp Thr Ser Thr PhePro Leu Glu Asn Val 210 215 220 ttg tcc atc ttc tta tac agt aat tca gactga 705 Leu Ser Ile Phe Leu Tyr Ser Asn Ser Asp 225 230 2 234 PRT Homosapiens 2 Met Asp Pro Gly Leu Gln Gln Ala Leu Asn Gly Met Ala Pro ProGly 1 5 10 15 Asp Thr Ala Met His Val Pro Ala Gly Ser Val Ala Ser HisLeu Gly 20 25 30 Thr Thr Ser Arg Ser Tyr Phe Tyr Leu Thr Thr Ala Thr LeuAla Leu 35 40 45 Cys Leu Val Phe Thr Val Ala Thr Ile Met Val Leu Val ValGln Arg 50 55 60 Thr Asp Ser Ile Pro Asn Ser Pro Asp Asn Val Pro Leu LysGly Gly 65 70 75 80 Asn Cys Ser Glu Asp Leu Leu Cys Ile Leu Lys Arg AlaPro Phe Lys 85 90 95 Lys Ser Trp Ala Tyr Leu Gln Val Ala Lys His Leu AsnLys Thr Lys 100 105 110 Leu Ser Trp Asn Lys Asp Gly Ile Leu His Gly ValArg Tyr Gln Asp 115 120 125 Gly Asn Leu Val Ile Gln Phe Pro Gly Leu TyrPhe Ile Ile Cys Gln 130 135 140 Leu Gln Phe Leu Val Gln Cys Pro Asn AsnSer Val Asp Leu Lys Leu 145 150 155 160 Glu Leu Leu Ile Asn Lys His IleLys Lys Gln Ala Leu Val Thr Val 165 170 175 Cys Glu Ser Gly Met Gln ThrLys His Val Tyr Gln Asn Leu Ser Gln 180 185 190 Phe Leu Leu Asp Tyr LeuGln Val Asn Thr Thr Ile Ser Val Asn Val 195 200 205 Asp Thr Phe Gln TyrIle Asp Thr Ser Thr Phe Pro Leu Glu Asn Val 210 215 220 Leu Ser Ile PheLeu Tyr Ser Asn Ser Asp 225 230 3 720 DNA Mus sp. CDS (1)..(720) 3 atggag cca ggg ctg caa caa gca ggc agc tgt ggg gct cct tcc cct 48 Met GluPro Gly Leu Gln Gln Ala Gly Ser Cys Gly Ala Pro Ser Pro 1 5 10 15 gaccca gcc atg cag gtg cag ccc ggc tcg gta gcc agc ccc tgg aga 96 Asp ProAla Met Gln Val Gln Pro Gly Ser Val Ala Ser Pro Trp Arg 20 25 30 agc acgagg ccc tgg aga agc aca agt cgc agc tac ttc tac ctc agc 144 Ser Thr ArgPro Trp Arg Ser Thr Ser Arg Ser Tyr Phe Tyr Leu Ser 35 40 45 acc acc gcactg gtg tgc ctt gtt gtg gca gtg gcg atc att ctg gta 192 Thr Thr Ala LeuVal Cys Leu Val Val Ala Val Ala Ile Ile Leu Val 50 55 60 ctg gta gtc cagaaa aag gac tcc act cca aat aca act gag aag gcc 240 Leu Val Val Gln LysLys Asp Ser Thr Pro Asn Thr Thr Glu Lys Ala 65 70 75 80 ccc ctt aaa ggagga aat tgc tca gag gat ctc ttc tgt acc ctg aaa 288 Pro Leu Lys Gly GlyAsn Cys Ser Glu Asp Leu Phe Cys Thr Leu Lys 85 90 95 agt act cca tcc aagaag tca tgg gcc tac ctc caa gtg tca aag cat 336 Ser Thr Pro Ser Lys LysSer Trp Ala Tyr Leu Gln Val Ser Lys His 100 105 110 ctc aac aat acc aaactg tca tgg aac gaa gat ggc acc atc cac gga 384 Leu Asn Asn Thr Lys LeuSer Trp Asn Glu Asp Gly Thr Ile His Gly 115 120 125 ctc ata tac cag gacggg aac ctg ata gtc caa ttc cct ggc ttg tac 432 Leu Ile Tyr Gln Asp GlyAsn Leu Ile Val Gln Phe Pro Gly Leu Tyr 130 135 140 ttc atc gtt tgc caactg cag ttc ctc gtg cag tgc tca aat cat tct 480 Phe Ile Val Cys Gln LeuGln Phe Leu Val Gln Cys Ser Asn His Ser 145 150 155 160 gtg gac ctg acattg cag ctc ctc atc aat tcc aag atc aaa aag cag 528 Val Asp Leu Thr LeuGln Leu Leu Ile Asn Ser Lys Ile Lys Lys Gln 165 170 175 acg ttg gta acagtg tgt gag tct gga gtt cag agt aag aac atc tac 576 Thr Leu Val Thr ValCys Glu Ser Gly Val Gln Ser Lys Asn Ile Tyr 180 185 190 cag aat ctc tctcag ttt ttg ctg cat tac tta cag gtc aac tct acc 624 Gln Asn Leu Ser GlnPhe Leu Leu His Tyr Leu Gln Val Asn Ser Thr 195 200 205 ata tca gtc agggtg gat aat ttc cag tat gtg gat aca aac act ttc 672 Ile Ser Val Arg ValAsp Asn Phe Gln Tyr Val Asp Thr Asn Thr Phe 210 215 220 cct ctt gat aatgtg cta tcc gtc ttc tta tat agt agc tca gac tga 720 Pro Leu Asp Asn ValLeu Ser Val Phe Leu Tyr Ser Ser Ser Asp 225 230 235 4 239 PRT Mus sp. 4Met Glu Pro Gly Leu Gln Gln Ala Gly Ser Cys Gly Ala Pro Ser Pro 1 5 1015 Asp Pro Ala Met Gln Val Gln Pro Gly Ser Val Ala Ser Pro Trp Arg 20 2530 Ser Thr Arg Pro Trp Arg Ser Thr Ser Arg Ser Tyr Phe Tyr Leu Ser 35 4045 Thr Thr Ala Leu Val Cys Leu Val Val Ala Val Ala Ile Ile Leu Val 50 5560 Leu Val Val Gln Lys Lys Asp Ser Thr Pro Asn Thr Thr Glu Lys Ala 65 7075 80 Pro Leu Lys Gly Gly Asn Cys Ser Glu Asp Leu Phe Cys Thr Leu Lys 8590 95 Ser Thr Pro Ser Lys Lys Ser Trp Ala Tyr Leu Gln Val Ser Lys His100 105 110 Leu Asn Asn Thr Lys Leu Ser Trp Asn Glu Asp Gly Thr Ile HisGly 115 120 125 Leu Ile Tyr Gln Asp Gly Asn Leu Ile Val Gln Phe Pro GlyLeu Tyr 130 135 140 Phe Ile Val Cys Gln Leu Gln Phe Leu Val Gln Cys SerAsn His Ser 145 150 155 160 Val Asp Leu Thr Leu Gln Leu Leu Ile Asn SerLys Ile Lys Lys Gln 165 170 175 Thr Leu Val Thr Val Cys Glu Ser Gly ValGln Ser Lys Asn Ile Tyr 180 185 190 Gln Asn Leu Ser Gln Phe Leu Leu HisTyr Leu Gln Val Asn Ser Thr 195 200 205 Ile Ser Val Arg Val Asp Asn PheGln Tyr Val Asp Thr Asn Thr Phe 210 215 220 Pro Leu Asp Asn Val Leu SerVal Phe Leu Tyr Ser Ser Ser Asp 225 230 235 5 1788 DNA Homo sapiens CDS(1)..(1788) 5 atg cgc gtc ctc ctc gcc gcg ctg gga ctg ctg ttc ctg ggggcg cta 48 Met Arg Val Leu Leu Ala Ala Leu Gly Leu Leu Phe Leu Gly AlaLeu 1 5 10 15 cga gcc ttc cca cag gat cga ccc ttc gag gac acc tgt catgga aac 96 Arg Ala Phe Pro Gln Asp Arg Pro Phe Glu Asp Thr Cys His GlyAsn 20 25 30 ccc agc cac tac tat gac aag gct gtc agg agg tgc tgt tac cgctgc 144 Pro Ser His Tyr Tyr Asp Lys Ala Val Arg Arg Cys Cys Tyr Arg Cys35 40 45 ccc atg ggg ctg ttc ccg aca cag cag tgc cca cag agg cct act gac192 Pro Met Gly Leu Phe Pro Thr Gln Gln Cys Pro Gln Arg Pro Thr Asp 5055 60 tgc agg aag cag tgt gag cct gac tac tac ctg gat gag gcc gac cgc240 Cys Arg Lys Gln Cys Glu Pro Asp Tyr Tyr Leu Asp Glu Ala Asp Arg 6570 75 80 tgt aca gcc tgc gtg act tgt tct cga gat gac ctc gtg gag aag acg288 Cys Thr Ala Cys Val Thr Cys Ser Arg Asp Asp Leu Val Glu Lys Thr 8590 95 ccg tgt gca tgg aac tcc tcc cgt gtc tgc gaa tgt cga ccc ggc atg336 Pro Cys Ala Trp Asn Ser Ser Arg Val Cys Glu Cys Arg Pro Gly Met 100105 110 ttc tgt tcc acg tct gcc gtc aac tcc tgt gcc cgc tgc ttc ttc cat384 Phe Cys Ser Thr Ser Ala Val Asn Ser Cys Ala Arg Cys Phe Phe His 115120 125 tct gtc tgt ccg gca ggg atg att gtc aag ttc cca ggc acg gcg cag432 Ser Val Cys Pro Ala Gly Met Ile Val Lys Phe Pro Gly Thr Ala Gln 130135 140 aag aac acg gtc tgt gag ccg gct tcc cca ggg gtc agc cct gcc tgt480 Lys Asn Thr Val Cys Glu Pro Ala Ser Pro Gly Val Ser Pro Ala Cys 145150 155 160 gcc agc cca gag aac tgc aag gaa ccc tcc agt ggc acc atc ccccag 528 Ala Ser Pro Glu Asn Cys Lys Glu Pro Ser Ser Gly Thr Ile Pro Gln165 170 175 gcc aag ccc acc ccg gtg tcc cca gca acc tcc agt gcc agc accatg 576 Ala Lys Pro Thr Pro Val Ser Pro Ala Thr Ser Ser Ala Ser Thr Met180 185 190 cct gta aga ggg ggc acc cgc ctc gcc cag gaa gct gct tct aaactg 624 Pro Val Arg Gly Gly Thr Arg Leu Ala Gln Glu Ala Ala Ser Lys Leu195 200 205 acg agg gct ccc gac tct ccc tcc tct gtg gga agg cct agt tcagat 672 Thr Arg Ala Pro Asp Ser Pro Ser Ser Val Gly Arg Pro Ser Ser Asp210 215 220 cca ggt ctg tcc cca aca cag cca tgc cca gag ggg tct ggt gattgc 720 Pro Gly Leu Ser Pro Thr Gln Pro Cys Pro Glu Gly Ser Gly Asp Cys225 230 235 240 aga aag cag tgt gag ccc gac tac tac ctg gac gag gcc ggccgc tgc 768 Arg Lys Gln Cys Glu Pro Asp Tyr Tyr Leu Asp Glu Ala Gly ArgCys 245 250 255 aca gcc tgc gtg agc tgt tct cga gat gac ctt gtg gag aagacg cca 816 Thr Ala Cys Val Ser Cys Ser Arg Asp Asp Leu Val Glu Lys ThrPro 260 265 270 tgt gca tgg aac tcc tcc cgc acc tgc gaa tgt cga cct ggcatg atc 864 Cys Ala Trp Asn Ser Ser Arg Thr Cys Glu Cys Arg Pro Gly MetIle 275 280 285 tgt gcc aca tca gcc acc aac tcc tgt gcc cgc tgt gtc ccctac cca 912 Cys Ala Thr Ser Ala Thr Asn Ser Cys Ala Arg Cys Val Pro TyrPro 290 295 300 atc tgt gca gga gag acg gtc acc aag ccc cag gat atg gctgag aag 960 Ile Cys Ala Gly Glu Thr Val Thr Lys Pro Gln Asp Met Ala GluLys 305 310 315 320 gac acc acc ttt gag gcg cca ccc ctg ggg acc cag ccggac tgc aac 1008 Asp Thr Thr Phe Glu Ala Pro Pro Leu Gly Thr Gln Pro AspCys Asn 325 330 335 ccc acc cca gag aat ggc gag gcg cct gcc agc acc agcccc act cag 1056 Pro Thr Pro Glu Asn Gly Glu Ala Pro Ala Ser Thr Ser ProThr Gln 340 345 350 agc ttg ctg gtg gac tcc cag gcc agt aag acg ctg cccatc cca acc 1104 Ser Leu Leu Val Asp Ser Gln Ala Ser Lys Thr Leu Pro IlePro Thr 355 360 365 agc gct ccc gtc gct ctc tcc tcc acg ggg aag ccc gttctg gat gca 1152 Ser Ala Pro Val Ala Leu Ser Ser Thr Gly Lys Pro Val LeuAsp Ala 370 375 380 ggg cca gtg ctc ttc tgg gtg atc ctg gtg ttg gtt gtggtg gtc ggc 1200 Gly Pro Val Leu Phe Trp Val Ile Leu Val Leu Val Val ValVal Gly 385 390 395 400 tcc agc gcc ttc ctc ctg tgc cac cgg agg gcc tgcagg aag cga att 1248 Ser Ser Ala Phe Leu Leu Cys His Arg Arg Ala Cys ArgLys Arg Ile 405 410 415 cgg cag aag ctc cac ctg tgc tac ccg gtc cag acctcc cag ccc aag 1296 Arg Gln Lys Leu His Leu Cys Tyr Pro Val Gln Thr SerGln Pro Lys 420 425 430 cta gag ctt gtg gat tcc aga ccc agg agg agc tcaacg cag ctg agg 1344 Leu Glu Leu Val Asp Ser Arg Pro Arg Arg Ser Ser ThrGln Leu Arg 435 440 445 agt ggt gcg tcg gtg aca gaa ccc gtc gcg gaa gagcga ggg tta atg 1392 Ser Gly Ala Ser Val Thr Glu Pro Val Ala Glu Glu ArgGly Leu Met 450 455 460 agc cag cca ctg atg gag acc tgc cac agc gtg ggggca gcc tac ctg 1440 Ser Gln Pro Leu Met Glu Thr Cys His Ser Val Gly AlaAla Tyr Leu 465 470 475 480 gag agc ctg ccg ctg cag gat gcc agc ccg gccggg ggc ccc tcg tcc 1488 Glu Ser Leu Pro Leu Gln Asp Ala Ser Pro Ala GlyGly Pro Ser Ser 485 490 495 ccc agg gac ctt cct gag ccc cgg gtg tcc acggag cac acc aat aac 1536 Pro Arg Asp Leu Pro Glu Pro Arg Val Ser Thr GluHis Thr Asn Asn 500 505 510 aag att gag aaa atc tac atc atg aag gct gacacc gtg atc gtg ggg 1584 Lys Ile Glu Lys Ile Tyr Ile Met Lys Ala Asp ThrVal Ile Val Gly 515 520 525 acc gtg aag gct gag ctg ccg gag ggc cgg ggcctg gcg ggg cca gca 1632 Thr Val Lys Ala Glu Leu Pro Glu Gly Arg Gly LeuAla Gly Pro Ala 530 535 540 gag ccc gag ttg gag gag gag ctg gag gcg gaccat acc ccc cac tac 1680 Glu Pro Glu Leu Glu Glu Glu Leu Glu Ala Asp HisThr Pro His Tyr 545 550 555 560 ccc gag cag gag aca gaa ccg cct ctg ggcagc tgc agc gat gtc atg 1728 Pro Glu Gln Glu Thr Glu Pro Pro Leu Gly SerCys Ser Asp Val Met 565 570 575 ctc tca gtg gaa gag gaa ggg aaa gaa gacccc ttg ccc aca gct gcc 1776 Leu Ser Val Glu Glu Glu Gly Lys Glu Asp ProLeu Pro Thr Ala Ala 580 585 590 tct gga aag tga 1788 Ser Gly Lys 595 6595 PRT Homo sapiens 6 Met Arg Val Leu Leu Ala Ala Leu Gly Leu Leu PheLeu Gly Ala Leu 1 5 10 15 Arg Ala Phe Pro Gln Asp Arg Pro Phe Glu AspThr Cys His Gly Asn 20 25 30 Pro Ser His Tyr Tyr Asp Lys Ala Val Arg ArgCys Cys Tyr Arg Cys 35 40 45 Pro Met Gly Leu Phe Pro Thr Gln Gln Cys ProGln Arg Pro Thr Asp 50 55 60 Cys Arg Lys Gln Cys Glu Pro Asp Tyr Tyr LeuAsp Glu Ala Asp Arg 65 70 75 80 Cys Thr Ala Cys Val Thr Cys Ser Arg AspAsp Leu Val Glu Lys Thr 85 90 95 Pro Cys Ala Trp Asn Ser Ser Arg Val CysGlu Cys Arg Pro Gly Met 100 105 110 Phe Cys Ser Thr Ser Ala Val Asn SerCys Ala Arg Cys Phe Phe His 115 120 125 Ser Val Cys Pro Ala Gly Met IleVal Lys Phe Pro Gly Thr Ala Gln 130 135 140 Lys Asn Thr Val Cys Glu ProAla Ser Pro Gly Val Ser Pro Ala Cys 145 150 155 160 Ala Ser Pro Glu AsnCys Lys Glu Pro Ser Ser Gly Thr Ile Pro Gln 165 170 175 Ala Lys Pro ThrPro Val Ser Pro Ala Thr Ser Ser Ala Ser Thr Met 180 185 190 Pro Val ArgGly Gly Thr Arg Leu Ala Gln Glu Ala Ala Ser Lys Leu 195 200 205 Thr ArgAla Pro Asp Ser Pro Ser Ser Val Gly Arg Pro Ser Ser Asp 210 215 220 ProGly Leu Ser Pro Thr Gln Pro Cys Pro Glu Gly Ser Gly Asp Cys 225 230 235240 Arg Lys Gln Cys Glu Pro Asp Tyr Tyr Leu Asp Glu Ala Gly Arg Cys 245250 255 Thr Ala Cys Val Ser Cys Ser Arg Asp Asp Leu Val Glu Lys Thr Pro260 265 270 Cys Ala Trp Asn Ser Ser Arg Thr Cys Glu Cys Arg Pro Gly MetIle 275 280 285 Cys Ala Thr Ser Ala Thr Asn Ser Cys Ala Arg Cys Val ProTyr Pro 290 295 300 Ile Cys Ala Gly Glu Thr Val Thr Lys Pro Gln Asp MetAla Glu Lys 305 310 315 320 Asp Thr Thr Phe Glu Ala Pro Pro Leu Gly ThrGln Pro Asp Cys Asn 325 330 335 Pro Thr Pro Glu Asn Gly Glu Ala Pro AlaSer Thr Ser Pro Thr Gln 340 345 350 Ser Leu Leu Val Asp Ser Gln Ala SerLys Thr Leu Pro Ile Pro Thr 355 360 365 Ser Ala Pro Val Ala Leu Ser SerThr Gly Lys Pro Val Leu Asp Ala 370 375 380 Gly Pro Val Leu Phe Trp ValIle Leu Val Leu Val Val Val Val Gly 385 390 395 400 Ser Ser Ala Phe LeuLeu Cys His Arg Arg Ala Cys Arg Lys Arg Ile 405 410 415 Arg Gln Lys LeuHis Leu Cys Tyr Pro Val Gln Thr Ser Gln Pro Lys 420 425 430 Leu Glu LeuVal Asp Ser Arg Pro Arg Arg Ser Ser Thr Gln Leu Arg 435 440 445 Ser GlyAla Ser Val Thr Glu Pro Val Ala Glu Glu Arg Gly Leu Met 450 455 460 SerGln Pro Leu Met Glu Thr Cys His Ser Val Gly Ala Ala Tyr Leu 465 470 475480 Glu Ser Leu Pro Leu Gln Asp Ala Ser Pro Ala Gly Gly Pro Ser Ser 485490 495 Pro Arg Asp Leu Pro Glu Pro Arg Val Ser Thr Glu His Thr Asn Asn500 505 510 Lys Ile Glu Lys Ile Tyr Ile Met Lys Ala Asp Thr Val Ile ValGly 515 520 525 Thr Val Lys Ala Glu Leu Pro Glu Gly Arg Gly Leu Ala GlyPro Ala 530 535 540 Glu Pro Glu Leu Glu Glu Glu Leu Glu Ala Asp His ThrPro His Tyr 545 550 555 560 Pro Glu Gln Glu Thr Glu Pro Pro Leu Gly SerCys Ser Asp Val Met 565 570 575 Leu Ser Val Glu Glu Glu Gly Lys Glu AspPro Leu Pro Thr Ala Ala 580 585 590 Ser Gly Lys 595 7 1357 DNA Homosapiens mat_peptide (193)..() 7 ctggaaaata cattctgcta ctcttaaaaactagtgacgc tcatacaaat caacagaaag 60 agcttctgaa ggaagacttt aaagctgcttctgccacgtg ctgctgggtc tcagtcctcc 120 acttcccgtg tcctctggaa gttgtcaggagca atg ttg cgc ttg tac gtg ttg 174 Met Leu Arg Leu Tyr Val Leu -10 gtaatg gga gtt tct gcc ttc acc ctt cag cct gcg gca cac aca ggg 222 Val MetGly Val Ser Ala Phe Thr Leu Gln Pro Ala Ala His Thr Gly -5 -1 1 5 10 gctgcc aga agc tgc cgg ttt cgt ggg agg cat tac aag cgg gag ttc 270 Ala AlaArg Ser Cys Arg Phe Arg Gly Arg His Tyr Lys Arg Glu Phe 15 20 25 agg ctggaa ggg gag cct gta gcc ctg agg tgc ccc cag gtg ccc tac 318 Arg Leu GluGly Glu Pro Val Ala Leu Arg Cys Pro Gln Val Pro Tyr 30 35 40 tgg ttg tgggcc tct gtc agc ccc cgc atc aac ctg aca tgg cat aaa 366 Trp Leu Trp AlaSer Val Ser Pro Arg Ile Asn Leu Thr Trp His Lys 45 50 55 aat gac tct gctagg acg gtc cca gga gaa gaa gag aca cgg atg tgg 414 Asn Asp Ser Ala ArgThr Val Pro Gly Glu Glu Glu Thr Arg Met Trp 60 65 70 gcc cag gac ggt gctctg tgg ctt ctg cca gcc ttg cag gag gac tct 462 Ala Gln Asp Gly Ala LeuTrp Leu Leu Pro Ala Leu Gln Glu Asp Ser 75 80 85 90 ggc acc tac gtc tgcact act aga aat gct tct tac tgt gac aaa atg 510 Gly Thr Tyr Val Cys ThrThr Arg Asn Ala Ser Tyr Cys Asp Lys Met 95 100 105 tcc att gag ctc agagtt ttt gag aat aca gat gct ttc ctg ccg ttc 558 Ser Ile Glu Leu Arg ValPhe Glu Asn Thr Asp Ala Phe Leu Pro Phe 110 115 120 atc tca tac ccg caaatt tta acc ttg tca acc tct ggg gta tta gta 606 Ile Ser Tyr Pro Gln IleLeu Thr Leu Ser Thr Ser Gly Val Leu Val 125 130 135 tgc cct gac ctg agtgaa ttc acc cgt gac aaa act gac gtg aag att 654 Cys Pro Asp Leu Ser GluPhe Thr Arg Asp Lys Thr Asp Val Lys Ile 140 145 150 caa tgg tac aag gattct ctt ctt ttg gat aaa gac aat gag aaa ttt 702 Gln Trp Tyr Lys Asp SerLeu Leu Leu Asp Lys Asp Asn Glu Lys Phe 155 160 165 170 cta agt gtg aggggg acc act cac tta ctc gta cac gat gtg gcc ctg 750 Leu Ser Val Arg GlyThr Thr His Leu Leu Val His Asp Val Ala Leu 175 180 185 gaa gat gct ggctat tac cgc tgt gtc ctg aca ttt gcc cat gaa ggc 798 Glu Asp Ala Gly TyrTyr Arg Cys Val Leu Thr Phe Ala His Glu Gly 190 195 200 cag caa tac aacatc act agg agt att gag cta cgc atc aag aaa aaa 846 Gln Gln Tyr Asn IleThr Arg Ser Ile Glu Leu Arg Ile Lys Lys Lys 205 210 215 aaa gaa gag accatt cct gtg atc att tcc ccc ctc aag acc ata tca 894 Lys Glu Glu Thr IlePro Val Ile Ile Ser Pro Leu Lys Thr Ile Ser 220 225 230 gct tct ctg gggtca aga ctg aca atc ccg tgt aag gtg ttt ctg gga 942 Ala Ser Leu Gly SerArg Leu Thr Ile Pro Cys Lys Val Phe Leu Gly 235 240 245 250 acc ggc acaccc tta acc acc atg ctg tgg tgg acg gcc aat gac acc 990 Thr Gly Thr ProLeu Thr Thr Met Leu Trp Trp Thr Ala Asn Asp Thr 255 260 265 cac ata gagagc gcc tac ccg gga ggc cgc gtg acc gag ggg cca cgc 1038 His Ile Glu SerAla Tyr Pro Gly Gly Arg Val Thr Glu Gly Pro Arg 270 275 280 cag gaa tattca gaa aat aat gag aac tac att gaa gtg cca ttg att 1086 Gln Glu Tyr SerGlu Asn Asn Glu Asn Tyr Ile Glu Val Pro Leu Ile 285 290 295 ttt gat cctgtc aca aga gag gat ttg cac atg gat ttt aaa tgt gtt 1134 Phe Asp Pro ValThr Arg Glu Asp Leu His Met Asp Phe Lys Cys Val 300 305 310 gtc cat aatacc ctg agt ttt cag aca cta cgc acc aca gtc aag gaa 1182 Val His Asn ThrLeu Ser Phe Gln Thr Leu Arg Thr Thr Val Lys Glu 315 320 325 330 gcc tcctcc acg ttc tcc tgg ggc att gtg ctg gcc cca ctt tca ctg 1230 Ala Ser SerThr Phe Ser Trp Gly Ile Val Leu Ala Pro Leu Ser Leu 335 340 345 gcc ttcttg gtt ttg ggg gga ata tgg atg cac aga cgg tgc aaa cac 1278 Ala Phe LeuVal Leu Gly Gly Ile Trp Met His Arg Arg Cys Lys His 350 355 360 aga actgga aaa gca gat ggt ctg act gtg cta tgg cct cat cat caa 1326 Arg Thr GlyLys Ala Asp Gly Leu Thr Val Leu Trp Pro His His Gln 365 370 375 gac tttcaa tcc tat ccc aag tgaaataaat 1357 Asp Phe Gln Ser Tyr Pro Lys 380 3858 398 PRT Homo sapiens 8 Met Leu Arg Leu Tyr Val Leu Val Met Gly Val SerAla Phe Thr Leu -10 -5 -1 1 Gln Pro Ala Ala His Thr Gly Ala Ala Arg SerCys Arg Phe Arg Gly 5 10 15 Arg His Tyr Lys Arg Glu Phe Arg Leu Glu GlyGlu Pro Val Ala Leu 20 25 30 35 Arg Cys Pro Gln Val Pro Tyr Trp Leu TrpAla Ser Val Ser Pro Arg 40 45 50 Ile Asn Leu Thr Trp His Lys Asn Asp SerAla Arg Thr Val Pro Gly 55 60 65 Glu Glu Glu Thr Arg Met Trp Ala Gln AspGly Ala Leu Trp Leu Leu 70 75 80 Pro Ala Leu Gln Glu Asp Ser Gly Thr TyrVal Cys Thr Thr Arg Asn 85 90 95 Ala Ser Tyr Cys Asp Lys Met Ser Ile GluLeu Arg Val Phe Glu Asn 100 105 110 115 Thr Asp Ala Phe Leu Pro Phe IleSer Tyr Pro Gln Ile Leu Thr Leu 120 125 130 Ser Thr Ser Gly Val Leu ValCys Pro Asp Leu Ser Glu Phe Thr Arg 135 140 145 Asp Lys Thr Asp Val LysIle Gln Trp Tyr Lys Asp Ser Leu Leu Leu 150 155 160 Asp Lys Asp Asn GluLys Phe Leu Ser Val Arg Gly Thr Thr His Leu 165 170 175 Leu Val His AspVal Ala Leu Glu Asp Ala Gly Tyr Tyr Arg Cys Val 180 185 190 195 Leu ThrPhe Ala His Glu Gly Gln Gln Tyr Asn Ile Thr Arg Ser Ile 200 205 210 GluLeu Arg Ile Lys Lys Lys Lys Glu Glu Thr Ile Pro Val Ile Ile 215 220 225Ser Pro Leu Lys Thr Ile Ser Ala Ser Leu Gly Ser Arg Leu Thr Ile 230 235240 Pro Cys Lys Val Phe Leu Gly Thr Gly Thr Pro Leu Thr Thr Met Leu 245250 255 Trp Trp Thr Ala Asn Asp Thr His Ile Glu Ser Ala Tyr Pro Gly Gly260 265 270 275 Arg Val Thr Glu Gly Pro Arg Gln Glu Tyr Ser Glu Asn AsnGlu Asn 280 285 290 Tyr Ile Glu Val Pro Leu Ile Phe Asp Pro Val Thr ArgGlu Asp Leu 295 300 305 His Met Asp Phe Lys Cys Val Val His Asn Thr LeuSer Phe Gln Thr 310 315 320 Leu Arg Thr Thr Val Lys Glu Ala Ser Ser ThrPhe Ser Trp Gly Ile 325 330 335 Val Leu Ala Pro Leu Ser Leu Ala Phe LeuVal Leu Gly Gly Ile Trp 340 345 350 355 Met His Arg Arg Cys Lys His ArgThr Gly Lys Ala Asp Gly Leu Thr 360 365 370 Val Leu Trp Pro His His GlnAsp Phe Gln Ser Tyr Pro Lys 375 380 385 9 23 DNA Artificial SequenceSynthetic primer 9 ggattgtcac ggtgccgttg aag 23 10 21 DNA ArtificialSequence Synthetic primer 10 ccggtggatg tggaatgtgt g 21 11 22 DNAArtificial Sequence Synthetic primer 11 tgctgatggg gatacatcca tc 22 1221 DNA Artificial Sequence Synthetic primer 12 ccggtggatg tggaatgtgt g21 13 20 DNA Artificial Sequence Synthetic primer 13 agagctccaggcacaagggc 20 14 19 DNA Artificial Sequence Synthetic primer 14aacgggccag acctcgggt 19 15 2478 DNA Homo sapiens CDS (1)..(2475) 15 atgggg tgg ctt tgc tct ggg ctc ctg ttc cct gtg agc tgc ctg gtc 48 Met GlyTrp Leu Cys Ser Gly Leu Leu Phe Pro Val Ser Cys Leu Val -25 -20 -15 -10ctg ctg cag gtg gca agc tct ggg aac atg aag gtc ttg cag gag ccc 96 LeuLeu Gln Val Ala Ser Ser Gly Asn Met Lys Val Leu Gln Glu Pro -5 -1 1 5acc tgc gtc tcc gac tac atg agc atc tct act tgc gag tgg aag atg 144 ThrCys Val Ser Asp Tyr Met Ser Ile Ser Thr Cys Glu Trp Lys Met 10 15 20 aatggt ccc acc aat tgc agc acc gag ctc cgc ctg ttg tac cag ctg 192 Asn GlyPro Thr Asn Cys Ser Thr Glu Leu Arg Leu Leu Tyr Gln Leu 25 30 35 gtt tttctg ctc tcc gaa gcc cac acg tgt atc cct gag aac aac gga 240 Val Phe LeuLeu Ser Glu Ala His Thr Cys Ile Pro Glu Asn Asn Gly 40 45 50 55 ggc gcgggg tgc gtg tgc cac ctg ctc atg gat gac gtg gtc agt gcg 288 Gly Ala GlyCys Val Cys His Leu Leu Met Asp Asp Val Val Ser Ala 60 65 70 gat aac tataca ctg gac ctg tgg gct ggg cag cag ctg ctg tgg aag 336 Asp Asn Tyr ThrLeu Asp Leu Trp Ala Gly Gln Gln Leu Leu Trp Lys 75 80 85 ggc tcc ttc aagccc agc gag cat gtg aaa ccc agg gcc cca gga aac 384 Gly Ser Phe Lys ProSer Glu His Val Lys Pro Arg Ala Pro Gly Asn 90 95 100 ctg aca gtt cacacc aat gtc tcc gac act ctg ctg ctg acc tgg agc 432 Leu Thr Val His ThrAsn Val Ser Asp Thr Leu Leu Leu Thr Trp Ser 105 110 115 aac ccg tat ccccct gac aat tac ctg tat aat cat ctc acc tat gca 480 Asn Pro Tyr Pro ProAsp Asn Tyr Leu Tyr Asn His Leu Thr Tyr Ala 120 125 130 135 gtc aac atttgg agt gaa aac gac ccg gca gat ttc aga atc tat aac 528 Val Asn Ile TrpSer Glu Asn Asp Pro Ala Asp Phe Arg Ile Tyr Asn 140 145 150 gtg acc taccta gaa ccc tcc ctc cgc atc gca gcc agc acc ctg aag 576 Val Thr Tyr LeuGlu Pro Ser Leu Arg Ile Ala Ala Ser Thr Leu Lys 155 160 165 tct ggg atttcc tac agg gca cgg gtg agg gcc tgg gct cag tgc tat 624 Ser Gly Ile SerTyr Arg Ala Arg Val Arg Ala Trp Ala Gln Cys Tyr 170 175 180 aac acc acctgg agt gag tgg agc ccc agc acc aag tgg cac aac tcc 672 Asn Thr Thr TrpSer Glu Trp Ser Pro Ser Thr Lys Trp His Asn Ser 185 190 195 tac agg gagccc ttc gag cag cac ctc ctg ctg ggc gtc agc gtt tcc 720 Tyr Arg Glu ProPhe Glu Gln His Leu Leu Leu Gly Val Ser Val Ser 200 205 210 215 tgc attgtc atc ctg gcc gtc tgc ctg ttg tgc tat gtc agc atc acc 768 Cys Ile ValIle Leu Ala Val Cys Leu Leu Cys Tyr Val Ser Ile Thr 220 225 230 aag attaag aaa gaa tgg tgg gat cag att ccc aac cca gcc cgc agc 816 Lys Ile LysLys Glu Trp Trp Asp Gln Ile Pro Asn Pro Ala Arg Ser 235 240 245 cgc ctcgtg gct ata ata atc cag gat gct cag ggg tca cag tgg gag 864 Arg Leu ValAla Ile Ile Ile Gln Asp Ala Gln Gly Ser Gln Trp Glu 250 255 260 aag cggtcc cga ggc cag gaa cca gcc aag tgc cca cac tgg aag aat 912 Lys Arg SerArg Gly Gln Glu Pro Ala Lys Cys Pro His Trp Lys Asn 265 270 275 tgt cttacc aag ctc ttg ccc tgt ttt ctg gag cac aac atg aaa agg 960 Cys Leu ThrLys Leu Leu Pro Cys Phe Leu Glu His Asn Met Lys Arg 280 285 290 295 gatgaa gat cct cac aag gct gcc aaa gag atg cct ttc cag ggc tct 1008 Asp GluAsp Pro His Lys Ala Ala Lys Glu Met Pro Phe Gln Gly Ser 300 305 310 ggaaaa tca gca tgg tgc cca gtg gag atc agc aag aca gtc ctc tgg 1056 Gly LysSer Ala Trp Cys Pro Val Glu Ile Ser Lys Thr Val Leu Trp 315 320 325 ccagag agc atc agc gtg gtg cga tgt gtg gag ttg ttt gag gcc ccg 1104 Pro GluSer Ile Ser Val Val Arg Cys Val Glu Leu Phe Glu Ala Pro 330 335 340 gtggag tgt gag gag gag gag gag gta gag gaa gaa aaa ggg agc ttc 1152 Val GluCys Glu Glu Glu Glu Glu Val Glu Glu Glu Lys Gly Ser Phe 345 350 355 tgtgca tcg cct gag agc agc agg gat gac ttc cag gag gga agg gag 1200 Cys AlaSer Pro Glu Ser Ser Arg Asp Asp Phe Gln Glu Gly Arg Glu 360 365 370 375ggc att gtg gcc cgg cta aca gag agc ctg ttc ctg gac ctg ctc gga 1248 GlyIle Val Ala Arg Leu Thr Glu Ser Leu Phe Leu Asp Leu Leu Gly 380 385 390gag gag aat ggg ggc ttt tgc cag cag gac atg ggg gag tca tgc ctt 1296 GluGlu Asn Gly Gly Phe Cys Gln Gln Asp Met Gly Glu Ser Cys Leu 395 400 405ctt cca cct tcg gga agt acg agt gct cac atg ccc tgg gat gag ttc 1344 LeuPro Pro Ser Gly Ser Thr Ser Ala His Met Pro Trp Asp Glu Phe 410 415 420cca agt gca ggg ccc aag gag gca cct ccc tgg ggc aag gag cag cct 1392 ProSer Ala Gly Pro Lys Glu Ala Pro Pro Trp Gly Lys Glu Gln Pro 425 430 435ctc cac ctg gag cca agt cct cct gcc agc ccg acc cag agt cca gac 1440 LeuHis Leu Glu Pro Ser Pro Pro Ala Ser Pro Thr Gln Ser Pro Asp 440 445 450455 aac ctg act tgc aca gag acg ccc ctc gtc atc gca ggc aac cct gct 1488Asn Leu Thr Cys Thr Glu Thr Pro Leu Val Ile Ala Gly Asn Pro Ala 460 465470 tac cgc agc ttc agc aac tcc ctg agc cag tca ccg tgt ccc aga gag 1536Tyr Arg Ser Phe Ser Asn Ser Leu Ser Gln Ser Pro Cys Pro Arg Glu 475 480485 ctg ggt cca gac cca ctg ctg gcc aga cac ctg gag gaa gta gaa ccc 1584Leu Gly Pro Asp Pro Leu Leu Ala Arg His Leu Glu Glu Val Glu Pro 490 495500 gag atg ccc tgt gtc ccc cag ctc tct gag cca acc act gtg ccc caa 1632Glu Met Pro Cys Val Pro Gln Leu Ser Glu Pro Thr Thr Val Pro Gln 505 510515 cct gag cca gaa acc tgg gag cag atc ctc cgc cga aat gtc ctc cag 1680Pro Glu Pro Glu Thr Trp Glu Gln Ile Leu Arg Arg Asn Val Leu Gln 520 525530 535 cat ggg gca gct gca gcc ccc gtc tcg gcc ccc acc agt ggc tat cag1728 His Gly Ala Ala Ala Ala Pro Val Ser Ala Pro Thr Ser Gly Tyr Gln 540545 550 gag ttt gta cat gcg gtg gag cag ggt ggc acc cag gcc agt gcg gtg1776 Glu Phe Val His Ala Val Glu Gln Gly Gly Thr Gln Ala Ser Ala Val 555560 565 gtg ggc ttg ggt ccc cca gga gag gct ggt tac aag gcc ttc tca agc1824 Val Gly Leu Gly Pro Pro Gly Glu Ala Gly Tyr Lys Ala Phe Ser Ser 570575 580 ctg ctt gcc agc agt gct gtg tcc cca gag aaa tgt ggg ttt ggg gct1872 Leu Leu Ala Ser Ser Ala Val Ser Pro Glu Lys Cys Gly Phe Gly Ala 585590 595 agc agt ggg gaa gag ggg tat aag cct ttc caa gac ctc att cct ggc1920 Ser Ser Gly Glu Glu Gly Tyr Lys Pro Phe Gln Asp Leu Ile Pro Gly 600605 610 615 tgc cct ggg gac cct gcc cca gtc cct gtc ccc ttg ttc acc tttgga 1968 Cys Pro Gly Asp Pro Ala Pro Val Pro Val Pro Leu Phe Thr Phe Gly620 625 630 ctg gac agg gag cca cct cgc agt ccg cag agc tca cat ctc ccaagc 2016 Leu Asp Arg Glu Pro Pro Arg Ser Pro Gln Ser Ser His Leu Pro Ser635 640 645 agc tcc cca gag cac ctg ggt ctg gag ccg ggg gaa aag gta gaggac 2064 Ser Ser Pro Glu His Leu Gly Leu Glu Pro Gly Glu Lys Val Glu Asp650 655 660 atg cca aag ccc cca ctt ccc cag gag cag gcc aca gac ccc cttgtg 2112 Met Pro Lys Pro Pro Leu Pro Gln Glu Gln Ala Thr Asp Pro Leu Val665 670 675 gac agc ctg ggc agt ggc att gtc tac tca gcc ctt acc tgc cacctg 2160 Asp Ser Leu Gly Ser Gly Ile Val Tyr Ser Ala Leu Thr Cys His Leu680 685 690 695 tgc ggc cac ctg aaa cag tgt cat ggc cag gag gat ggt ggccag acc 2208 Cys Gly His Leu Lys Gln Cys His Gly Gln Glu Asp Gly Gly GlnThr 700 705 710 cct gtc atg gcc agt cct tgc tgt ggc tgc tgc tgt gga gacagg tcc 2256 Pro Val Met Ala Ser Pro Cys Cys Gly Cys Cys Cys Gly Asp ArgSer 715 720 725 tcg ccc cct aca acc ccc ctg agg gcc cca gac ccc tct ccaggt ggg 2304 Ser Pro Pro Thr Thr Pro Leu Arg Ala Pro Asp Pro Ser Pro GlyGly 730 735 740 gtt cca ctg gag gcc agt ctg tgt ccg gcc tcc ctg gca ccctcg ggc 2352 Val Pro Leu Glu Ala Ser Leu Cys Pro Ala Ser Leu Ala Pro SerGly 745 750 755 atc tca gag aag agt aaa tcc tca tca tcc ttc cat cct gcccct ggc 2400 Ile Ser Glu Lys Ser Lys Ser Ser Ser Ser Phe His Pro Ala ProGly 760 765 770 775 aat gct cag agc tca agc cag acc ccc aaa atc gtg aacttt gtc tcc 2448 Asn Ala Gln Ser Ser Ser Gln Thr Pro Lys Ile Val Asn PheVal Ser 780 785 790 gtg gga ccc aca tac atg agg gtc tct tat 2478 Val GlyPro Thr Tyr Met Arg Val Ser 795 800 16 825 PRT Homo sapiens 16 Met GlyTrp Leu Cys Ser Gly Leu Leu Phe Pro Val Ser Cys Leu Val -25 -20 -15 -10Leu Leu Gln Val Ala Ser Ser Gly Asn Met Lys Val Leu Gln Glu Pro -5 -1 15 Thr Cys Val Ser Asp Tyr Met Ser Ile Ser Thr Cys Glu Trp Lys Met 10 1520 Asn Gly Pro Thr Asn Cys Ser Thr Glu Leu Arg Leu Leu Tyr Gln Leu 25 3035 Val Phe Leu Leu Ser Glu Ala His Thr Cys Ile Pro Glu Asn Asn Gly 40 4550 55 Gly Ala Gly Cys Val Cys His Leu Leu Met Asp Asp Val Val Ser Ala 6065 70 Asp Asn Tyr Thr Leu Asp Leu Trp Ala Gly Gln Gln Leu Leu Trp Lys 7580 85 Gly Ser Phe Lys Pro Ser Glu His Val Lys Pro Arg Ala Pro Gly Asn 9095 100 Leu Thr Val His Thr Asn Val Ser Asp Thr Leu Leu Leu Thr Trp Ser105 110 115 Asn Pro Tyr Pro Pro Asp Asn Tyr Leu Tyr Asn His Leu Thr TyrAla 120 125 130 135 Val Asn Ile Trp Ser Glu Asn Asp Pro Ala Asp Phe ArgIle Tyr Asn 140 145 150 Val Thr Tyr Leu Glu Pro Ser Leu Arg Ile Ala AlaSer Thr Leu Lys 155 160 165 Ser Gly Ile Ser Tyr Arg Ala Arg Val Arg AlaTrp Ala Gln Cys Tyr 170 175 180 Asn Thr Thr Trp Ser Glu Trp Ser Pro SerThr Lys Trp His Asn Ser 185 190 195 Tyr Arg Glu Pro Phe Glu Gln His LeuLeu Leu Gly Val Ser Val Ser 200 205 210 215 Cys Ile Val Ile Leu Ala ValCys Leu Leu Cys Tyr Val Ser Ile Thr 220 225 230 Lys Ile Lys Lys Glu TrpTrp Asp Gln Ile Pro Asn Pro Ala Arg Ser 235 240 245 Arg Leu Val Ala IleIle Ile Gln Asp Ala Gln Gly Ser Gln Trp Glu 250 255 260 Lys Arg Ser ArgGly Gln Glu Pro Ala Lys Cys Pro His Trp Lys Asn 265 270 275 Cys Leu ThrLys Leu Leu Pro Cys Phe Leu Glu His Asn Met Lys Arg 280 285 290 295 AspGlu Asp Pro His Lys Ala Ala Lys Glu Met Pro Phe Gln Gly Ser 300 305 310Gly Lys Ser Ala Trp Cys Pro Val Glu Ile Ser Lys Thr Val Leu Trp 315 320325 Pro Glu Ser Ile Ser Val Val Arg Cys Val Glu Leu Phe Glu Ala Pro 330335 340 Val Glu Cys Glu Glu Glu Glu Glu Val Glu Glu Glu Lys Gly Ser Phe345 350 355 Cys Ala Ser Pro Glu Ser Ser Arg Asp Asp Phe Gln Glu Gly ArgGlu 360 365 370 375 Gly Ile Val Ala Arg Leu Thr Glu Ser Leu Phe Leu AspLeu Leu Gly 380 385 390 Glu Glu Asn Gly Gly Phe Cys Gln Gln Asp Met GlyGlu Ser Cys Leu 395 400 405 Leu Pro Pro Ser Gly Ser Thr Ser Ala His MetPro Trp Asp Glu Phe 410 415 420 Pro Ser Ala Gly Pro Lys Glu Ala Pro ProTrp Gly Lys Glu Gln Pro 425 430 435 Leu His Leu Glu Pro Ser Pro Pro AlaSer Pro Thr Gln Ser Pro Asp 440 445 450 455 Asn Leu Thr Cys Thr Glu ThrPro Leu Val Ile Ala Gly Asn Pro Ala 460 465 470 Tyr Arg Ser Phe Ser AsnSer Leu Ser Gln Ser Pro Cys Pro Arg Glu 475 480 485 Leu Gly Pro Asp ProLeu Leu Ala Arg His Leu Glu Glu Val Glu Pro 490 495 500 Glu Met Pro CysVal Pro Gln Leu Ser Glu Pro Thr Thr Val Pro Gln 505 510 515 Pro Glu ProGlu Thr Trp Glu Gln Ile Leu Arg Arg Asn Val Leu Gln 520 525 530 535 HisGly Ala Ala Ala Ala Pro Val Ser Ala Pro Thr Ser Gly Tyr Gln 540 545 550Glu Phe Val His Ala Val Glu Gln Gly Gly Thr Gln Ala Ser Ala Val 555 560565 Val Gly Leu Gly Pro Pro Gly Glu Ala Gly Tyr Lys Ala Phe Ser Ser 570575 580 Leu Leu Ala Ser Ser Ala Val Ser Pro Glu Lys Cys Gly Phe Gly Ala585 590 595 Ser Ser Gly Glu Glu Gly Tyr Lys Pro Phe Gln Asp Leu Ile ProGly 600 605 610 615 Cys Pro Gly Asp Pro Ala Pro Val Pro Val Pro Leu PheThr Phe Gly 620 625 630 Leu Asp Arg Glu Pro Pro Arg Ser Pro Gln Ser SerHis Leu Pro Ser 635 640 645 Ser Ser Pro Glu His Leu Gly Leu Glu Pro GlyGlu Lys Val Glu Asp 650 655 660 Met Pro Lys Pro Pro Leu Pro Gln Glu GlnAla Thr Asp Pro Leu Val 665 670 675 Asp Ser Leu Gly Ser Gly Ile Val TyrSer Ala Leu Thr Cys His Leu 680 685 690 695 Cys Gly His Leu Lys Gln CysHis Gly Gln Glu Asp Gly Gly Gln Thr 700 705 710 Pro Val Met Ala Ser ProCys Cys Gly Cys Cys Cys Gly Asp Arg Ser 715 720 725 Ser Pro Pro Thr ThrPro Leu Arg Ala Pro Asp Pro Ser Pro Gly Gly 730 735 740 Val Pro Leu GluAla Ser Leu Cys Pro Ala Ser Leu Ala Pro Ser Gly 745 750 755 Ile Ser GluLys Ser Lys Ser Ser Ser Ser Phe His Pro Ala Pro Gly 760 765 770 775 AsnAla Gln Ser Ser Ser Gln Thr Pro Lys Ile Val Asn Phe Val Ser 780 785 790Val Gly Pro Thr Tyr Met Arg Val Ser 795 800

What is claimed is:
 1. A method of treating an autoimmune or chronicinflammatory condition in a patient, said method comprisingadministering to the patient an antibody specific for CD30L that iscapable of inhibiting the binding of CD30 to CD30L, wherein the antibodyis administered according to a regimen of dose and frequency ofadministration that is adequate to induce a sustained improvement in atleast one indicator that reflects the severity of the patient'scondition, the improvement being considered sustained if the patientexhibits the improvement on at least two occasions separated by at leastone day and further wherein the patient's condition is selected from thegroup consisting of psoriatic arthritis; arthritis nodosa; seronegativespondylarthropathies, and inflammatory bowel diseases.
 2. The methodaccording to claim 1, wherein the patient is a human.
 3. The methodaccording to claim 2, wherein the condition is ankylosing spondylitis.4. The method according to claim 2, wherein the condition is psoriaticarthritis.
 5. The method according to claim 2, wherein the condition isselected from the group consisting of Crohn's disease and ulcerativecolitis.
 6. The method according to claim 2, wherein the antibody isadministered concurrently with a second agent that is an antagonist ofTNFα, IL-1α, IL-1β or IL-4.
 7. The method according to claim 6, whereinthe second agent is an antagonist of TNFα selected from the groupconsisting of etanercept, p55 TNFR-Ig fusion protein and an antibodyagainst TNFα.
 8. The method according to claim 7, wherein the antagonistof TNFα is an antibody against TNFα, and further wherein said antibodyis selected from the group consisting of infliximab, D2E7 and CDP571. 9.The method according to claim 2, wherein the antibody specific for CD30Lis a monoclonal antibody.
 10. The method according to claim 9, whereinthe antibody specific for CD30L is a humanized antibody.
 11. The methodaccording to claim 9, wherein the antibody specific for CD30L is a humanantibody.
 12. The method according to claim 3, wherein the antibodyspecific for CD30L is a monoclonal antibody.
 13. The method according toclaim 12, wherein the antibody specific for CD30L is a humanizedantibody.
 14. The method according to claim 12, wherein the antibodyspecific for CD30L is a human antibody.
 15. The method according toclaim 4, wherein the antibody specific for CD30L is a monoclonalantibody.
 16. The method according to claim 15, wherein the antibodyspecific for CD30L is a humanized antibody.
 17. The method according toclaim 15, wherein the antibody specific for CD30L is a human antibody.18. The method according to claim 5, wherein the antibody specific forCD30L is a monoclonal antibody.
 19. The method according to claim 18,wherein the antibody specific for CD30L is a humanized antibody.
 20. Themethod according to claim 18, wherein the antibody specific for CD30L isa human antibody.
 21. The method according to claim 6, wherein theantibody specific for CD30L is a monoclonal antibody.
 22. The methodaccording to claim 21, wherein the antibody specific for CD30L is ahumanized antibody.
 23. The method according to claim 21, wherein theantibody specific for CD30L is a human antibody.
 24. An animal model forscreening therapeutic agents, said animal model being characterized by:(a) carrying genetic modifications that inactivate its p55 and p75 TNFαreceptor proteins; and (b) being genetically susceptible toexperimentally-induced arthritis.
 25. The animal model according toclaim 24, wherein the arthritis to which the animal model is geneticallysusceptible is collagen-induced arthritis.
 26. The animal modelaccording to claim 25, wherein the animal model is a strain of mousethat is selected from the group consisting of DBA/1, BUB and B10.Q or astrain of rat that is selected from the group consisting of DA, BB-DRand LEW.
 27. The animal model according to claim 26, wherein the strainof mouse is DBA/1, and further wherein the DBA/1 strain of mouse hasdouble-null mutations in its p55 and p75 TNFα receptor genes.
 28. Amethod for screening a candidate therapeutic agent to determine itsefficacy in treating an autoimmune or chronic inflammatory conditionthat is resistant to treatment with a TNFα inhibitor, said methodcomprising inducing arthritis in the animal model of claim 27,administering the candidate therapeutic agent to said animal, anddetermining that the agent is efficacious if the severity of saidanimal's arthritis is reduced after the candidate agent has beenadministered.